Cargo-loading of hybrid cowpea chlorotic mottle virus capsids via a co-expression approach.

Capsids of the cowpea chlorotic mottle virus (CCMV) are great candidates for the development into in vivo catalytic or therapeutic nanocarriers. However, due to their limited intrinsic stability at physiological pH, thus far no methods exist for incorporating cargo into these nanoparticles in cellulo. Here, we employ a stabilized VW1-VW8 ELP-CCMV variant for the development of a co-expression-based cargo-loading approach. Co-expression of the non-functionalized VW1-VW8 ELP-CCMV coat protein with fusion proteins with enhanced green fluorescent protein (mEGFP) and pyrrolysine synthase D (PylD) in E. coli enabled the purification of cargo-loaded capsids from the bacteria directly either via affinity chromatography or PEG-precipitation and subsequent size exclusion chromatography. Microscopy results indicated that the co-expression does not harm the E. coli cells and that proper folding of the mEGFP domain is not hampered by the co-assembly. Our co-expression strategy is thus a suitable approach to produce cargo-loaded CCMV nanoparticles.

Effect of temperature on the compositions of ladderane lipids in globally surveyed anammox populations.

The adaptation of bacteria involved in anaerobic ammonium oxidation (anammox) to low temperatures will enable more efficient removal of nitrogen from sewage across seasons. At lower temperatures, bacteria typically tune the synthesis of their membrane lipids to promote membrane fluidity. However, such adaptation of anammox bacteria lipids, including unique ladderane phospholipids and especially shorter ladderanes with absent phosphatidyl headgroup, is yet to be described in detail. We investigated the membrane lipids composition (UPLC-HRMS/MS) and dominant anammox populations (16S rRNA gene amplicon sequencing, Fluorescence in situ hybridization) in 14 anammox enrichments cultivated at 10-37 °C. "Candidatus Brocadia" appeared to be the dominant organism in all but two laboratory enrichments of "Ca. Scalindua" and "Ca. Kuenenia". At lower temperatures, the membranes of all anammox populations were composed of shorter [5]-ladderane ester (reduced chain length demonstrated by decreased fraction of C20/(C18 + C20)). This confirmed the previous preliminary evidence on the prominent role of this ladderane fatty acid in low-temperature adaptation. "Ca. Scalindua" and "Ca. Kuenenia" had distinct profile of ladderane lipids compared to "Ca. Brocadia" biomasses with potential implications for adaptability to low temperatures. "Ca. Brocadia" membranes contained a much lower amount of C18 [5]-ladderane esters than reported in the literature for "Ca. Scalindua" at similar temperature and measured here, suggesting that this could be one of the reasons for the dominance of "Ca. Scalindua" in cold marine environments. Furthermore, we propose additional and yet unreported mechanisms for low-temperature adaptation of anammox bacteria, one of which involves ladderanes with absent phosphatidyl headgroup. In sum, we deepen the understanding of cold anammox physiology by providing for the first time a consistent comparison of anammox-based communities across multiple environments.

Endocarditis Caused by Nontypeable Streptococcus pneumoniae.

The Streptococcus pneumoniae capsule is regarded as indispensable in bacteremia. We report an infant with a ventricular septal defect and infective endocarditis caused by nontypeable S. pneumoniae. In-depth investigation confirmed a deficient capsule yet favored pneumococcal fitness for causing infective endocarditis, rather than a host immune disorder, as the cause of infective endocarditis in this case.

Antimicrobial Late Cornified Envelope Proteins: The Psoriasis Risk Factor Deletion of LCE3B/C Genes Affects Microbiota Composition.

Late cornified envelope proteins are predominantly expressed in the skin and other cornified epithelia. On the basis of sequence similarity, this 18-member homologous gene family has been subdivided into six groups. The LCE3 proteins have been the focus of dermatological research because the combined deletion of LCE3B and LCE3C genes (LCE3B/C-del) is a risk factor for psoriasis. We previously reported that LCE3B/C-del increases the expression of the LCE3A gene and that LCE3 proteins exert antibacterial activity. In this study, we analyzed the antimicrobial properties of other family members and the role of LCE3B/C-del in the modulation of microbiota composition of the skin and oral cavity. Differences in killing efficiency and specificity between the late cornified envelope proteins and their target microbes were found, and the amino acid content rather than the order of the well-conserved central domain of the LCE3A protein was found responsible for its antibacterial activity. In vivo, LCE3B/C-del correlated with a higher beta-diversity in the skin and oral microbiota. From these results, we conclude that all late cornified envelope proteins possess antimicrobial activity. Tissue-specific and genotype-dependent antimicrobial protein profiles impact skin and oral microbiota composition, which could direct toward LCE3B/C-del‒associated dysbiosis and a possible role for microbiota in the pathophysiology of psoriasis.

The Polygonal Cell Shape and Surface Protein Layer of Anaerobic Methane-Oxidizing Methylomirabilis lanthanidiphila Bacteria.

Methylomirabilis bacteria perform anaerobic methane oxidation coupled to nitrite reduction via an intra-aerobic pathway, producing carbon dioxide and dinitrogen gas. These diderm bacteria possess an unusual polygonal cell shape with sharp ridges that run along the cell body. Previously, a putative surface protein layer (S-layer) was observed as the outermost cell layer of these bacteria. We hypothesized that this S-layer is the determining factor for their polygonal cell shape. Therefore, we enriched the S-layer from M. lanthanidiphila cells and through LC-MS/MS identified a 31 kDa candidate S-layer protein, mela_00855, which had no homology to any other known protein. Antibodies were generated against a synthesized peptide derived from the mela_00855 protein sequence and used in immunogold localization to verify its identity and location. Both on thin sections of M. lanthanidiphila cells and in negative-stained enriched S-layer patches, the immunogold localization identified mela_00855 as the S-layer protein. Using electron cryo-tomography and sub-tomogram averaging of S-layer patches, we observed that the S-layer has a hexagonal symmetry. Cryo-tomography of whole cells showed that the S-layer and the outer membrane, but not the peptidoglycan layer and the cytoplasmic membrane, exhibited the polygonal shape. Moreover, the S-layer consisted of multiple rigid sheets that partially overlapped, most likely giving rise to the unique polygonal cell shape. These characteristics make the S-layer of M. lanthanidiphila a distinctive and intriguing case to study.

Structural and functional characterization of the intracellular filament-forming nitrite oxidoreductase multiprotein complex.

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.

Multiheme hydroxylamine oxidoreductases produce NO during ammonia oxidation in methanotrophs.

Aerobic and nitrite-dependent methanotrophs make a living from oxidizing methane via methanol to carbon dioxide. In addition, these microorganisms cometabolize ammonia due to its structural similarities to methane. The first step in both of these processes is catalyzed by methane monooxygenase, which converts methane or ammonia into methanol or hydroxylamine, respectively. Methanotrophs use methanol for energy conservation, whereas toxic hydroxylamine is a potent inhibitor that needs to be rapidly removed. It is suggested that many methanotrophs encode a hydroxylamine oxidoreductase (mHAO) in their genome to remove hydroxylamine, although biochemical evidence for this is lacking. HAOs also play a crucial role in the metabolism of aerobic and anaerobic ammonia oxidizers by converting hydroxylamine to nitric oxide (NO). Here, we purified an HAO from the thermophilic verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV and characterized its kinetic properties. This mHAO possesses the characteristic P460 chromophore and is active up to at least 80 °C. It catalyzes the rapid oxidation of hydroxylamine to NO. In methanotrophs, mHAO efficiently removes hydroxylamine, which severely inhibits calcium-dependent, and as we show here, lanthanide-dependent methanol dehydrogenases, which are more prevalent in the environment. Our results indicate that mHAO allows methanotrophs to thrive under high ammonia concentrations in natural and engineered ecosystems, such as those observed in rice paddy fields, landfills, or volcanic mud pots, by preventing the accumulation of inhibitory hydroxylamine. Under oxic conditions, methanotrophs mainly oxidize ammonia to nitrite, whereas in hypoxic and anoxic environments reduction of both ammonia-derived nitrite and NO could lead to nitrous oxide (N2O) production.

Nutrient Limitation Causes Differential Expression of Transport- and Metabolism Genes in the Compartmentalized Anammox Bacterium Kuenenia stuttgartiensis.

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.

Growth on Carbohydrates from Carbonaceous Meteorites Alters the Immunogenicity of Environment-Derived Bacterial Pathogens.

The last decade has witnessed a renewed interest in space exploration. Public and private institutions are investing considerable effort toward the direct exploration of the Moon and Mars, as well as more distant bodies in the solar system. Both automated and human-crewed spacecraft are being considered in these efforts. As inevitable fellow travelers on the bodies of astronauts, spaceships, or equipment, terrestrial microorganisms will undoubtedly come into contact with extraterrestrial environments, despite stringent decontamination. These microorganisms could eventually adapt and grow in their new habitats, where they might potentially recolonize and lead to the infection of the human space travelers. In this article, we demonstrate that clinically relevant bacterial species found in the environment are able to grow in minimal media with sugar compounds identified in extraterrestrial carbon sources. As a surrogate model, we used carbohydrates previously isolated from carbonaceous meteorites. The bacteria underwent an adaptation process that caused structural modifications in the cell envelope that sparked changes in pathogenic potential, both in vitro and in vivo. Understanding the adaptation of microorganisms exposed to extraterrestrial environments, with subsequent changes in their immunogenicity and virulence, requires a comprehensive analysis of such scenarios to ensure the safety of major space expeditions in the decades to come.

Extracellular electron transfer-dependent anaerobic oxidation of ammonium by anammox bacteria.

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.

Non-essentiality of canonical cell division genes in the planctomycete Planctopirus limnophila.

Most bacteria divide by binary fission using an FtsZ-based mechanism that relies on a multi-protein complex, the divisome. In the majority of non-spherical bacteria another multi-protein complex, the elongasome, is also required for the maintenance of cell shape. Components of these multi-protein assemblies are conserved and essential in most bacteria. Here, we provide evidence that at least three proteins of these two complexes are not essential in the FtsZ-less ovoid planctomycete bacterium Planctopirus limnophila which divides by budding. We attempted to construct P. limnophila knock-out mutants of the genes coding for the divisome proteins FtsI, FtsK, FtsW and the elongasome protein MreB. Surprisingly, ftsI, ftsW and mreB could be deleted without affecting the growth rate. On the other hand, the conserved ftsK appeared to be essential in this bacterium. In conclusion, the canonical bacterial cell division machinery is not essential in P. limnophila and this bacterium divides via budding using an unknown mechanism.

Complexome analysis of the nitrite-dependent methanotroph Methylomirabilis lanthanidiphila.

The atmospheric concentration of the potent greenhouse gases methane and nitrous oxide (N2O) has increased drastically during the last century. Methylomirabilis bacteria can play an important role in controlling the emission of these two gases from natural ecosystems, by oxidizing methane to CO2 and reducing nitrite to N2 without producing N2O. These bacteria have an anaerobic metabolism, but are proposed to possess an oxygen-dependent pathway for methane activation. Methylomirabilis bacteria reduce nitrite to NO, and are proposed to dismutate NO into O2 and N2 by a putative NO dismutase (NO-D). The O2 produced in the cell can then be used to activate methane by a particulate methane monooxygenase. So far, the metabolic model of Methylomirabilis bacteria was based mainly on (meta)genomics and physiological experiments. Here we applied a complexome profiling approach to determine which of the proposed enzymes are actually expressed in Methylomirabilis lanthanidiphila. To validate the proposed metabolic model, we focused on enzymes involved in respiration, as well as nitrogen and carbon transformation. All complexes suggested to be involved in nitrite-dependent methane oxidation, were identified in M. lanthanidiphila, including the putative NO-D. Furthermore, several complexes involved in nitrate reduction/nitrite oxidation and NO reduction were detected, which likely play a role in detoxification and redox homeostasis. In conclusion, complexome profiling validated the expression and composition of enzymes hypothesized to be involved in the energy, methane and nitrogen metabolism of M. lanthanidiphila, thereby further corroborating their unique metabolism involved in the environmentally relevant process of nitrite-dependent methane oxidation.

Characterization of a novel cytochrome cGJ as the electron acceptor of XoxF-MDH in the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV.

Methanotrophs play a prominent role in the global carbon cycle, by oxidizing the potent greenhouse gas methane to CO2. Methane is first converted into methanol by methane monooxygenase. This methanol is subsequently oxidized by either a calcium-dependent MxaF-type or a lanthanide-dependent XoxF-type methanol dehydrogenase (MDH). Electrons from methanol oxidation are shuttled to a cytochrome redox partner, termed cytochrome cL. Here, the cytochrome cL homolog from the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV was characterized. SolV cytochrome cGJ is a fusion of a XoxG cytochrome and a periplasmic binding protein XoxJ. Here we show that XoxGJ functions as the direct electron acceptor of its corresponding XoxF-type MDH and can sustain methanol turnover, when a secondary cytochrome is present as final electron acceptor. SolV cytochrome cGJ (XoxGJ) further displays a unique, red-shifted absorbance spectrum, with a Soret and Q bands at 440, 553 and 595 nm in the reduced state, respectively. VTVH-MCD spectroscopy revealed the presence of a low spin iron heme and the data further shows that the heme group exhibits minimal ruffling. The midpoint potential Em,pH7 of +240 mV is similar to other cytochrome cL type proteins but remarkably, the midpoint potential of cytochrome cGJ was not influenced by lowering the pH. Cytochrome cGJ represents the first example of a cytochrome from a strictly lanthanide-dependent methylotrophic microorganism.

Trending topics and open questions in anaerobic ammonium oxidation.

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.

Bioreactor virome metagenomics sequencing using DNA spike-ins.

With the emergence of Next Generation Sequencing, major advances were made with regard to identifying viruses in natural environments. However, bioinformatical research on viruses is still limited because of the low amounts of viral DNA that can be obtained for analysis. To overcome this limitation, DNA is often amplified with multiple displacement amplification (MDA), which may cause an unavoidable bias. Here, we describe a case study in which the virome of a bioreactor is sequenced using Ion Torrent technology. DNA-spiking of samples is compared with MDA-amplified samples. DNA for spiking was obtained by amplifying a bacterial 16S rRNA gene. After sequencing, the 16S rRNA gene reads were removed by mapping to the Silva database. Three samples were tested, a whole genome from Enterobacteria P1 Phage and two viral metagenomes from an infected bioreactor. For one sample, the new DNA-spiking protocol was compared with the MDA technique. When MDA was applied, the overall GC content of the reads showed a bias towards lower GC%, indicating a change in composition of the DNA sample. Assemblies using all available reads from both MDA and the DNA-spiked samples resulted in six viral genomes. All six genomes could be almost completely retrieved (97.9%-100%) when mapping the reads from the DNA-spiked sample to those six genomes. In contrast, 6.3%-77.7% of three viral genomes was covered by reads obtained using the MDA amplification method and only three were nearly fully covered (97.4%-100%). This case study shows that DNA-spiking could be a simple and inexpensive alternative with very low bias for sequencing of metagenomes for which low amounts of DNA are available.

Characterization of the first planctomycetal outer membrane protein identifies a channel in the outer membrane of the anammox bacterium Kuenenia stuttgartiensis.

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 ß-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.

Community Composition and Ultrastructure of a Nitrate-Dependent Anaerobic Methane-Oxidizing Enrichment Culture.

Methane is a very potent greenhouse gas and can be oxidized aerobically or anaerobically through microbe-mediated processes, thus decreasing methane emissions in the atmosphere. Using a complementary array of methods, including phylogenetic analysis, physiological experiments, and light and electron microscopy techniques (including electron tomography), we investigated the community composition and ultrastructure of a continuous bioreactor enrichment culture, in which anaerobic oxidation of methane (AOM) was coupled to nitrate reduction. A membrane bioreactor was seeded with AOM biomass and continuously fed with excess methane. After 150 days, the bioreactor reached a daily consumption of 10 mmol nitrate · liter-1 · day-1 The biomass consisted of aggregates that were dominated by nitrate-dependent anaerobic methane-oxidizing "Candidatus Methanoperedens"-like archaea (40%) and nitrite-dependent anaerobic methane-oxidizing "Candidatus Methylomirabilis"-like bacteria (50%). The "Ca Methanoperedens" spp. were identified by fluorescence in situ hybridization and immunogold localization of the methyl-coenzyme M reductase (Mcr) enzyme, which was located in the cytoplasm. The "Ca Methanoperedens" sp. aggregates consisted of slightly irregular coccoid cells (∼1.5-µm diameter) which produced extruding tubular structures and putative cell-to-cell contacts among each other. "Ca Methylomirabilis" sp. bacteria exhibited the polygonal cell shape typical of this genus. In AOM archaea and bacteria, cytochrome c proteins were localized in the cytoplasm and periplasm, respectively, by cytochrome staining. Our results indicate that AOM bacteria and archaea might work closely together in the process of anaerobic methane oxidation, as the bacteria depend on the archaea for nitrite. Future studies will be aimed at elucidating the function of the cell-to-cell interactions in nitrate-dependent AOM.IMPORTANCE Microorganisms performing nitrate- and nitrite-dependent anaerobic methane oxidation are important in both natural and man-made ecosystems, such as wastewater treatment plants. In both systems, complex microbial interactions take place that are largely unknown. Revealing these microbial interactions would enable us to understand how the oxidation of the important greenhouse gas methane occurs in nature and pave the way for the application of these microbes in wastewater treatment plants. Here, we elucidated the microbial composition, ultrastructure, and physiology of a nitrate-dependent AOM community of archaea and bacteria and describe the cell plan of "Ca Methanoperedens"-like methanotrophic archaea.

Determining the bacterial cell biology of Planctomycetes.

Bacteria of the phylum Planctomycetes have been previously reported to possess several features that are typical of eukaryotes, such as cytosolic compartmentalization and endocytosis-like macromolecule uptake. However, recent evidence points towards a Gram-negative cell plan for Planctomycetes, although in-depth experimental analysis has been hampered by insufficient genetic tools. Here we develop methods for expression of fluorescent proteins and for gene deletion in a model planctomycete, Planctopirus limnophila, to analyse its cell organization in detail. Super-resolution light microscopy of mutants, cryo-electron tomography, bioinformatic predictions and proteomic analyses support an altered Gram-negative cell plan for Planctomycetes, including a defined outer membrane, a periplasmic space that can be greatly enlarged and convoluted, and an energized cytoplasmic membrane. These conclusions are further supported by experiments performed with two other Planctomycetes, Gemmata obscuriglobus and Rhodopirellula baltica. We also provide experimental evidence that is inconsistent with endocytosis-like macromolecule uptake; instead, extracellular macromolecules can be taken up and accumulate in the periplasmic space through unclear mechanisms.

The S-Layer Protein of the Anammox Bacterium Kuenenia stuttgartiensis Is Heavily O-Glycosylated.

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.