Structure of the two-component S-layer of the archaeon Sulfolobus acidocaldarius.

Surface layers (S-layers) are resilient two-dimensional protein lattices that encapsulate many bacteria and most archaea. In archaea, S-layers usually form the only structural component of the cell wall and thus act as the final frontier between the cell and its environment. Therefore, S-layers are crucial for supporting microbial life. Notwithstanding their importance, little is known about archaeal S-layers at the atomic level. Here, we combined single-particle cryo electron microscopy, cryo electron tomography, and Alphafold2 predictions to generate an atomic model of the two-component S-layer of Sulfolobus acidocaldarius. The outer component of this S-layer (SlaA) is a flexible, highly glycosylated, and stable protein. Together with the inner and membrane-bound component (SlaB), they assemble into a porous and interwoven lattice. We hypothesise that jackknife-like conformational changes in SlaA play important roles in S-layer assembly.

CryoEM reveals that ribosomes in microsporidian spores are locked in a dimeric hibernating state.

Translational control is an essential process for the cell to adapt to varying physiological or environmental conditions. To survive adverse conditions such as low nutrient levels, translation can be shut down almost entirely by inhibiting ribosomal function. Here we investigated eukaryotic hibernating ribosomes from the microsporidian parasite Spraguea lophii in situ by a combination of electron cryo-tomography and single-particle electron cryo-microscopy. We show that microsporidian spores contain hibernating ribosomes that are locked in a dimeric (100S) state, which is formed by a unique dimerization mechanism involving the beak region. The ribosomes within the dimer are fully assembled, suggesting that they are ready to be activated once the host cell is invaded. This study provides structural evidence for dimerization acting as a mechanism for ribosomal hibernation in microsporidia, and therefore demonstrates that eukaryotes utilize this mechanism in translational control.

Escaping the symmetry trap in helical reconstruction.

Helical reconstruction is the method of choice for obtaining 3D structures of filaments from electron cryo-microscopy (cryoEM) projections. This approach relies on applying helical symmetry parameters deduced from Fourier-Bessel or real space analysis, such as sub-tomogram averaging. While helical reconstruction continues to provide invaluable structural insights into filaments, its inherent dependence on imposing a pre-defined helical symmetry can also introduce bias. The applied helical symmetry produces structures that are infinitely straight along the filament's axis and can average out biologically important heterogeneities. Here, we describe a simple workflow aimed at overcoming these drawbacks in order to provide truer representations of filamentous structures.

An archaellum filament composed of two alternating subunits.

Archaea use a molecular machine, called the archaellum, to swim. The archaellum consists of an ATP-powered intracellular motor that drives the rotation of an extracellular filament composed of multiple copies of proteins named archaellins. In many species, several archaellin homologs are encoded in the same operon; however, previous structural studies indicated that archaellum filaments mainly consist of only one protein species. Here, we use electron cryo-microscopy to elucidate the structure of the archaellum from Methanocaldococcus villosus at 3.08 Å resolution. The filament is composed of two alternating archaellins, suggesting that the architecture and assembly of archaella is more complex than previously thought. Moreover, we identify structural elements that may contribute to the filament's flexibility.

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.

Architecture and modular assembly of Sulfolobus S-layers revealed by electron cryotomography.

Surface protein layers (S-layers) often form the only structural component of the archaeal cell wall and are therefore important for cell survival. S-layers have a plethora of cellular functions including maintenance of cell shape, osmotic, and mechanical stability, the formation of a semipermeable protective barrier around the cell, and cell-cell interaction, as well as surface adhesion. Despite the central importance of S-layers for archaeal life, their 3-dimensional (3D) architecture is still poorly understood. Here we present detailed 3D electron cryomicroscopy maps of archaeal S-layers from 3 different Sulfolobus strains. We were able to pinpoint the positions and determine the structure of the 2 subunits SlaA and SlaB. We also present a model describing the assembly of the mature S-layer.

Comparative Genomics of Candidatus Methylomirabilis Species and Description of Ca. Methylomirabilis Lanthanidiphila.

Methane is a potent greenhouse gas, which can be converted by microorganism at the expense of oxygen, nitrate, nitrite, metal-oxides or sulfate. The bacterium 'Candidatus Methylomirabilis oxyfera,' a member of the NC10 phylum, is capable of nitrite-dependent anaerobic methane oxidation. Prolonged enrichment of 'Ca. M. oxyfera' with cerium added as trace element and without nitrate resulted in the shift of the dominant species. Here, we present a high quality draft genome of the new species 'Candidatus Methylomirabilis lanthanidiphila' and use comparative genomics to analyze its metabolic potential in both nitrogen and carbon cycling. To distinguish between gene content specific for the 'Ca. Methylomirabilis' genus and the NC10 phylum, the genome of a distantly related NC10 phylum member, CSP1-5, an aerobic methylotroph, is included in the analysis. All genes for the conversion of nitrite to N2 identified in 'Ca. M. oxyfera' are conserved in 'Ca. M. lanthanidiphila,' including the two putative genes for NO dismutase. In addition both species have several heme-copper oxidases potentially involved in NO and O2 respiration. For the oxidation of methane 'Ca. Methylomirabilis' species encode a membrane bound methane monooxygenase. CSP1-5 can act as a methylotroph, but lacks the ability to activate methane. In contrast to 'Ca. M. oxyfera,' which harbors three methanol dehydrogenases (MDH), both CSP1-5 and 'Ca. M. lanthanidiphila' only encode a lanthanide-dependent XoxF-type MDH, once more underlining the importance of rare earth elements for methylotrophic bacteria. The pathways for the subsequent oxidation of formaldehyde to carbon dioxide and for the Calvin-Benson-Bassham cycle are conserved in all species. Furthermore, CSP1-5 can only interconvert nitrate and nitrite, but lacks subsequent nitrite or NO reductases. Thus, it appears that although the conversion of methanol to carbon dioxide is present in several NC10 phylum bacteria, the coupling of nitrite reduction to the oxidation of methane is a trait so far unique to the genus 'Ca. Methylomirabilis.'

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.

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.

Ultrastructure and Viral Metagenome of Bacteriophages from an Anaerobic Methane Oxidizing Methylomirabilis Bioreactor Enrichment Culture.

With its capacity for anaerobic methane oxidation and denitrification, the bacterium Methylomirabilis oxyfera plays an important role in natural ecosystems. Its unique physiology can be exploited for more sustainable wastewater treatment technologies. However, operational stability of full-scale bioreactors can experience setbacks due to, for example, bacteriophage blooms. By shaping microbial communities through mortality, horizontal gene transfer, and metabolic reprogramming, bacteriophages are important players in most ecosystems. Here, we analyzed an infected Methylomirabilis sp. bioreactor enrichment culture using (advanced) electron microscopy, viral metagenomics and bioinformatics. Electron micrographs revealed four different viral morphotypes, one of which was observed to infect Methylomirabilis cells. The infected cells contained densely packed ~55 nm icosahedral bacteriophage particles with a putative internal membrane. Various stages of virion assembly were observed. Moreover, during the bacteriophage replication, the host cytoplasmic membrane appeared extremely patchy, which suggests that the bacteriophages may use host bacterial lipids to build their own putative internal membrane. The viral metagenome contained 1.87 million base pairs of assembled viral sequences, from which five putative complete viral genomes were assembled and manually annotated. Using bioinformatics analyses, we could not identify which viral genome belonged to the Methylomirabilis- infecting bacteriophage, in part because the obtained viral genome sequences were novel and unique to this reactor system. Taken together these results show that new bacteriophages can be detected in anaerobic cultivation systems and that the effect of bacteriophages on the microbial community in these systems is a topic for further study.