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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.

The archaellum is a molecular machine used by archaea to swim, consisting of an intracellular motor that drives the rotation of an extracellular filament composed of multiple copies of proteins named archaellins. Here, the authors use electron cryo-microscopy to elucidate the structure of an archaellum, and find that the filament is composed of two alternating archaellins.

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.

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.’

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.

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 analysed 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.