Molecular SARS-CoV-2 surveillance in Bavaria shows no Omicron transmission before the end of November 2021.

BACKGROUND: Five SARS-CoV-2 variants are currently considered as variants of concern (VOC). Omicron was declared a VOC at the end of November 2021. Based on different diagnostic methods, the occurrence of Omicron was reported by 52 countries worldwide on December 7 2021. First notified by South Africa with alarming reports on increasing infection rates, this new variant was soon suspected to replace the currently pre-dominating Delta variant leading to further infection waves worldwide. METHODS: Using VOC PCR screening and Next Generation Sequencing (NGS) analysis of selected samples, we investigated the circulation of Omicron in the German federal state Bavaria. For this, we analyzed SARS-CoV-2 surveillance data from our laboratory generated from calendar week (CW) 01 to 49/2021. RESULTS: So far, we have detected 69 Omicron cases in our laboratory from CW 47-49/2021 using RT-qPCR followed by melting curve analysis. The first 16 cases were analyzed by NGS and all were confirmed as Omicron. CONCLUSION: Our data strongly support no circulation of the new Omicron variant before CW 47/2021.

Functional compartmentalization and metabolic separation in a prokaryotic cell.

The prokaryotic cell is traditionally seen as a "bag of enzymes," yet its organization is much more complex than in this simplified view. By now, various microcompartments encapsulating metabolic enzymes or pathways are known for Bacteria These microcompartments are usually small, encapsulating and concentrating only a few enzymes, thus protecting the cell from toxic intermediates or preventing unwanted side reactions. The hyperthermophilic, strictly anaerobic Crenarchaeon Ignicoccus hospitalis is an extraordinary organism possessing two membranes, an inner and an energized outer membrane. The outer membrane (termed here outer cytoplasmic membrane) harbors enzymes involved in proton gradient generation and ATP synthesis. These two membranes are separated by an intermembrane compartment, whose function is unknown. Major information processes like DNA replication, RNA synthesis, and protein biosynthesis are located inside the "cytoplasm" or central cytoplasmic compartment. Here, we show by immunogold labeling of ultrathin sections that enzymes involved in autotrophic CO2 assimilation are located in the intermembrane compartment that we name (now) a peripheric cytoplasmic compartment. This separation may protect DNA and RNA from reactive aldehydes arising in the I. hospitalis carbon metabolism. This compartmentalization of metabolic pathways and information processes is unprecedented in the prokaryotic world, representing a unique example of spatiofunctional compartmentalization in the second domain of life.

Reciprocal circulation pattern of SARS-CoV-2 and influenza viruses during the influenza seasons 2019/2020 and 2020/2021 in the Bavarian Influenza Sentinel (Germany).

The corona virus disease-2019 (COVID-19) pandemic began in Wuhan, China, and quickly spread around the world. The pandemic overlapped with two consecutive influenza seasons (2019/2020 and 2020/2021). This provided the opportunity to study community circulation of influenza viruses and severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) in outpatients with acute respiratory infections during these two seasons within the Bavarian Influenza Sentinel (BIS) in Bavaria, Germany. From September to March, oropharyngeal swabs collected at BIS were analysed for influenza viruses and SARS-CoV-2 by real-time polymerase chain reaction. In BIS 2019/2020, 1376 swabs were tested for influenza viruses. The average positive rate was 37.6%, with a maximum of over 60% (in January). The predominant influenza viruses were Influenza A(H1N1)pdm09 (n = 202), Influenza A(H3N2) (n = 144) and Influenza B Victoria lineage (n = 129). In all, 610 of these BIS swabs contained sufficient material to retrospectively test for SARS-CoV-2. SARS-CoV-2 RNA was not detectable in any of these swabs. In BIS 2020/2021, 470 swabs were tested for influenza viruses and 457 for SARS-CoV-2. Only three swabs (0.6%) were positive for Influenza, while SARS-CoV-2 was found in 30 swabs (6.6%). We showed that no circulation of SARS-CoV-2 was detectable in BIS during the 2019/2020 influenza season, while virtually no influenza viruses were found in BIS 2020/2021 during the COVID-19 pandemic.

2D and 3D immunogold localization on (epoxy) ultrathin sections with and without osmium tetroxide.

For nearly 50 years immunogold labeling on ultrathin sections has been successfully used for protein localization in laboratories worldwide. In theory and in practice, this method has undergone continual improvement over time. In this study, we carefully analyzed circulating protocols for postembedding labeling to find out if they are still valid under modern laboratory conditions, and in addition, we tested unconventional protocols. For this, we investigated immunolabeling of Epon-embedded cells, immunolabeling of cells treated with osmium, and the binding behavior of differently sized gold particles. Here we show that (in contrast to widespread belief) immunolabeling of Epon-embedded cells and of cells treated with osmium tetroxide is actually working. Furthermore, we established a "speed protocol" for immunolabeling by reducing antibody incubation times. Finally, we present our results on three-dimensional immunogold labeling.

Archaeal Cell Walls.

The cell wall of archaea, as of any other prokaryote, is surrounding the cell outside the cytoplasmic membrane and is mediating the interaction with the environment. In this regard, it can be involved in cell shape maintenance, protection against virus, heat, acidity or alkalinity. Throughout the formation of pore like structures, it can resemble a micro sieve and thereby enable or disable transport processes. In some cases, cell wall components can make up more than 10% of the whole cellular protein. So far, a great variety of different cell envelope structures and compounds have be found and described in detail. From all archaeal cell walls described so far, the most common structure is the S-layer. Other archaeal cell wall structures are pseudomurein, methanochondroitin, glutaminylglycan, sulfated heteropolysaccharides and protein sheaths and they are sometimes associated with additional proteins and protein complexes like the STABLE protease or the bindosome. Recent advances in electron microscopy also illustrated the presence of an outer(most) cellular membrane within several archaeal groups, comparable to the Gram-negative cell wall within bacteria. Each new cell wall structure that can be investigated in detail and that can be assigned with a specific function helps us to understand, how the earliest cells on earth might have looked like.

One-megadalton metalloenzyme complex in Geobacter metallireducens involved in benzene ring reduction beyond the biological redox window.

Reversible biological electron transfer usually occurs between redox couples at standard redox potentials ranging from +0.8 to -0.5 V. Dearomatizing benzoyl-CoA reductases (BCRs), key enzymes of the globally relevant microbial degradation of aromatic compounds at anoxic sites, catalyze a biological Birch reduction beyond the negative limit of this redox window. The structurally characterized BamBC subunits of class II BCRs accomplish benzene ring reduction at an active-site tungsten cofactor; however, the mechanism and components involved in the energetic coupling of endergonic benzene ring reduction have remained hypothetical. We present a 1-MDa, membrane-associated, Bam[(BC)2DEFGHI]2 complex from the anaerobic bacterium Geobacter metallireducens harboring 4 tungsten, 4 zinc, 2 selenocysteines, 6 FAD, and >50 FeS cofactors. The results suggest that class II BCRs catalyze electron transfer to the aromatic ring, yielding a cyclic 1,5-dienoyl-CoA via two flavin-based electron bifurcation events. This work expands our knowledge of energetic couplings in biology by high-molecular-mass electron bifurcating machineries.

Outer Membrane Proteome of Veillonella parvula: A Diderm Firmicute of the Human Microbiome.

Veillonella parvula is a biofilm-forming commensal found in the lungs, vagina, mouth, and gastro-intestinal tract of humans, yet it may develop into an opportunistic pathogen. Furthermore, the presence of Veillonella has been associated with the development of a healthy immune system in infants. Veillonella belongs to the Negativicutes, a diverse clade of bacteria that represent an evolutionary enigma: they phylogenetically belong to Gram-positive (monoderm) Firmicutes yet maintain an outer membrane (OM) with lipopolysaccharide similar to classic Gram-negative (diderm) bacteria. The OMs of Negativicutes have unique characteristics including the replacement of Braun's lipoprotein by OmpM for tethering the OM to the peptidoglycan. Through phylogenomic analysis, we have recently provided bioinformatic annotation of the Negativicutes diderm cell envelope. We showed that it is a unique type of envelope that was present in the ancestor of present-day Firmicutes and lost multiple times independently in this phylum, giving rise to the monoderm architecture; however, little experimental data is presently available for any Negativicutes cell envelope. Here, we performed the first experimental proteomic characterization of the cell envelope of a diderm Firmicute, producing an OM proteome of V. parvula. We initially conducted a thorough bioinformatics analysis of all 1,844 predicted proteins from V. parvula DSM 2008's genome using 12 different localization prediction programs. These results were complemented by protein extraction with surface exposed (SE) protein tags and by subcellular fractionation, both of which were analyzed by liquid chromatography tandem mass spectrometry. The merging of proteomics and bioinformatics results allowed identification of 78 OM proteins. These include a number of receptors for TonB-dependent transport, the main component of the BAM system for OM protein biogenesis (BamA), the Lpt system component LptD, which is responsible for insertion of LPS into the OM, and several copies of the major OmpM protein. The annotation of V. parvula's OM proteome markedly extends previous inferences on the nature of the cell envelope of Negativicutes, including the experimental evidence of a BAM/TAM system for OM protein biogenesis and of a complete Lpt system for LPS transport to the OM. It also provides important information on the role of OM components in the lifestyle of Veillonella, such as a possible gene cluster for O-antigen synthesis and a large number of adhesins. Finally, many OM hypothetical proteins were identified, which are priority targets for further characterization.

A Complex Endomembrane System in the Archaeon Ignicoccus hospitalis Tapped by Nanoarchaeum equitans.

Based on serial sectioning, focused ion beam scanning electron microscopy (FIB/SEM), and electron tomography, we depict in detail the highly unusual anatomy of the marine hyperthermophilic crenarchaeon, Ignicoccus hospitalis. Our data support a complex and dynamic endomembrane system consisting of cytoplasmic protrusions, and with secretory function. Moreover, we reveal that the cytoplasm of the putative archaeal ectoparasite Nanoarchaeum equitans can get in direct contact with this endomembrane system, complementing and explaining recent proteomic, transcriptomic and metabolomic data on this inter-archaeal relationship. In addition, we identified a matrix of filamentous structures and/or tethers in the voluminous inter-membrane compartment (IMC) of I. hospitalis, which might be responsible for membrane dynamics. Overall, this unusual cellular compartmentalization, ultrastructure and dynamics in an archaeon that belongs to the recently proposed TACK superphylum prompts speculation that the eukaryotic endomembrane system might originate from Archaea.

Cytochromes c in Archaea: distribution, maturation, cell architecture, and the special case of Ignicoccus hospitalis.

Cytochromes c (Cytc) are widespread electron transfer proteins and important enzymes in the global nitrogen and sulfur cycles. The distribution of Cytc in more than 300 archaeal proteomes deduced from sequence was analyzed with computational methods including pattern and similarity searches, secondary and tertiary structure prediction. Two hundred and fifty-eight predicted Cytc (with single, double, or multiple heme c attachment sites) were found in some but not all species of the Desulfurococcales, Thermoproteales, Archaeoglobales, Methanosarcinales, Halobacteriales, and in two single-cell genome sequences of the Thermoplasmatales, all of them Cren- or Euryarchaeota. Other archaeal phyla including the Thaumarchaeota are so far free of these proteins. The archaeal Cytc sequences were bundled into 54 clusters of mutual similarity, some of which were specific for Archaea while others had homologs in the Bacteria. The cytochrome c maturation system I (CCM) was the only one found. The highest number and variability of Cytc were present in those species with known or predicted metal oxidation and/or reduction capabilities. Paradoxical findings were made in the haloarchaea: several Cytc had been purified biochemically but corresponding proteins were not found in the proteomes. The results are discussed with emphasis on cell morphologies and envelopes and especially for double-membraned Archaea-like Ignicoccus hospitalis. A comparison is made with compartmentalized bacteria such as the Planctomycetes of the Anammox group with a focus on the putative localization and roles of the Cytc and other electron transport proteins.

Three multihaem cytochromes c from the hyperthermophilic archaeon Ignicoccus hospitalis: purification, properties and localization.

Three different multihaem cytochromes c were purified from cell extracts of the hyperthermophilic archaeon Ignicoccus hospitalis. One tetrahaem cytochrome, locus tag designation Igni_0530, was purified from membrane fractions together with the iron-sulfur protein Igni_0529. Two octahaem cytochromes, Igni_0955 and Igni_1359, were purified from soluble fractions but were also present in the membrane fraction. N-terminal sequencing showed that three of the four proteins had their signal peptides cleaved off, while results were ambiguous for Igni_0955. In contrast, mass spectrometry of Igni_0955 and Igni_1359 resulted in single mass peaks including the signal sequences and eight haems per subunit and so both forms might be present in the cell. Igni_0955 and Igni_1359 belong to the hydroxylamine dehydrogenase (HAO) family (29 % mutual identity). HAO or reductase activities with inorganic sulfur compounds were not detected. Igni_0955 was reduced by enriched I. hospitalis hydrogenase at a specific activity of 243 nmol min(-1) (mg hydrogenase)(-1) while activity was non-existent for Igni_0530 and low for Igni_1359. Immuno-electron microscopy of ultra-thin sections showed that Igni_0955 and Igni_1359 are located in both I. hospitalis membranes and also in the intermembrane compartment. We concluded that these cytochromes might function as electron shuttles between the hydrogenase in the outer cellular membrane and cellular reductases, whereas Igni_0530 might be part of the sulfur-reducing mechanism.