Isolation of Resistance-Bearing Microorganisms

To better exploit the principles of gas transport and mass transport during the processes of cell seeding of 3D scaffolds and in vitro culture of 3D tissue engineered constructs, the oscillatory cell culture bioreactor provides a flow of cell suspensions and culture media directly through a porous 3D scaffold (during cell seeding) and a 3D construct (during subsequent cultivation) within a highly gas-permeable closed-loop tube. This design is simple, modular, and flexible, and its component parts are easy to assemble and operate, and are inexpensive. Chamber volume can be very low, but can be easily scaled up. This innovation is well suited to work with different biological specimens, particularly with cells having high oxygen requirements and/or shear sensitivity, and different scaffold structures and dimensions. The closed-loop changer is highly gas permeable to allow efficient gas exchange during the cell seeding/culturing process. A porous scaffold, which may be seeded with cells, is fixed by means of a scaffold holder to the chamber wall with scaffold/construct orientation with respect to the chamber determined by the geometry of the scaffold holder. A fluid, with/without biological specimens, is added to the chamber such that all, or most, of the air is displaced (i.e., with or without an enclosed air bubble). Motion is applied to the chamber within a controlled environment (e.g., oscillatory motion within a humidified 37 C incubator). Movement of the chamber induces relative motion of the scaffold/construct with respect to the fluid. In case the fluid is a cell suspension, cells will come into contact with the scaffold and eventually adhere to it. Alternatively, cells can be seeded on scaffolds by gel entrapment prior to bioreactor cultivation. Subsequently, the oscillatory cell culture bioreactor will provide efficient gas exchange (i.e., of oxygen and carbon dioxide, as required for viability of metabolically active cells) and controlled levels of fluid dynamic shear (i.e., as required for viability of shear-sensitive cells) to the developing engineered tissue construct. This bioreactor was recently utilized to show independent and interactive effects of a growth factor (IGF-I) and slow bidirectional perfusion on the survival, differentiation, and contractile performance of 3D tissue engineering cardiac constructs. The main application of this system is within the tissue engineering industry. The ideal final application is within the automated mass production of tissue- engineered constructs. Target industries could be both life sciences companies as well as bioreactor device producing companies

Overlooked Diversity of Ultramicrobacterial Minorities at the Air-Sea Interface

Members of the Candidate phylum Patescibacteria, also called Candidate Phyla Radiation (CPR), are described as ultramicrobacteria with limited metabolic capacities. Wide diversity and relative abundances up to 80% in anaerobic habitats, e.g., in groundwater or sediments are characteristic for Candidatus Patescibacteria. However, only few studies exist for marine surface water. Here, we report the presence of 40 patescibacterial candidate clades at air-sea interfaces, including the upper water layer, floating foams and the sea-surface microlayer (SML), a < 1 mm layer at the boundary between ocean and atmosphere. Particle-associated (>3 μm) and free-living (3–0.2 μm) samples were obtained from the Jade Bay, North Sea, and 16S rRNA (gene) amplicons were analyzed. Although the abundance of Cand. Patescibacteria representatives were relatively low (<1.3%), members of Cand. Kaiserbacteria and Cand. Gracilibacteria were found in all samples. This suggests profound aerotolerant capacities of these phylogenetic lineages at the air-sea interface. The presence of ultramicrobacteria in the >3 μm fraction implies adhesion to bigger aggregates, potentially in anoxic niches, and a symbiotic lifestyle. Due to their small sizes, Cand. Patescibacteria likely become aerosolized to the atmosphere and dispersed to land with possible implications for affecting microbial communities and associated processes in these ecosystems.J.R.: C.S., O.W. and this study were funded by the European Research Council project PASSME, grant number GA336408. The picture of seafoam was taken during a campaign funded by the Assemble Plus project MIDSEAS (European Union’s Horizon 2020 research and innovation program, Grant Agreement No. 730984). D.P.R.H. was supported by the European Regional Development Fund/Estonian Research Council funded by “Mobilitas Plus Top Researcher grant MOBTT24”. A.J.P. received funding by the Ministerium für Kultur und Wissenschaft des Landes Nordrhein-Westfalen (“Nachwuchsgruppe Alexander Probst”). The APC was funded by the Open Access Publication Fund of the University of Duisburg-Essen.J.R.: C.S., O.W. and this study were funded by the European Research Council project PASSME, grant number GA336408. The picture of seafoam was taken during a campaign funded by the Assemble Plus project MIDSEAS (European Union’s Horizon 2020 research and innovation program, Grant Agreement No. 730984). D.P.R.H. was supported by the European Regional Development Fund/Estonian Research Council funded by “Mobilitas Plus Top Researcher grant MOBTT24”. A.J.P. received funding by the Ministerium für Kultur und Wissenschaft des Landes Nordrhein-Westfalen (“Nachwuchsgruppe Alexander Probst”). The APC was funded by the Open Access Publication Fund of the University of Duisburg-Essen

Urban Dust Microbiome: Impact on Later Atopy and Wheezing

INTRODUCTION: Investigations in urban areas have just begun to explore how the indoor dust microbiome may affect the pathogenesis of asthma and allery. We aimed to investigate the early fungal and bacterial microbiome in house dust with allergic sensitization and wheezing later in childhood. METHODS: Individual dust samples from 189 homes of the LISAplus birth cohort study were collected shortly after birth from living room floors and profiled for fungal and bacterial microbiome. Fungal and bacterial diversity was assessed with terminal restriction fragment length polymorphism (tRFLP) and defined by the Simpson diversity index. Information on wheezing outcomes and co-variates until the age of 10 years was obtained by parental questionnaires. Information on specific allergic sensitization was available at 6 and 10 years. Logistic regression and General Estimation Equation (GEE) models were used to examine the relationship between microbial diversity and health outcomes. RESULTS: Logistic regression analyses revealed a significantly reduced risk of developing sensitization to aero-allergens at 6 years and ever wheezing until the age of 10 years for exposure to higher fungal diversity (adjusted Odds Ratio aOR (95%CI): 0.26 (0.10-0.70)), and 0.42 (0.18-0.96), respectively), in adjusted analyses. The associations were attenuated for the longitudinal analyses (GEE) until the age of 10 years. There was no association between higher exposure to bacterial diversity and the tested health outcomes. CONCLUSION: Higher early exposure to fungal diversity might help to prevent from developing sensitization to aero-allergens in early childhood, but the reasons for attenuated effects in later childhood require further prospective studies

Microbial Community Structures of Novel Icelandic Hot Spring Systems Revealed by PhyloChip G3 Analysis

Microbial community profiles of recently formed hot spring systems ranging in temperatures from 57°C to 100°C and pH values from 2 to 4 in Hveragerði (Iceland) were analyzed with PhyloChip G3 technology. In total, 1173 bacterial operational taxonomic units (OTUs) spanning 576 subfamilies and 38 archaeal OTUs covering 32 subfamilies were observed. As expected, the hyperthermophilic (100°C) spring system exhibited both low microbial biomass and diversity when compared to thermophilic (60°C) springs. Ordination analysis revealed distinct bacterial and archaeal diversity in geographically distinct hot springs. Slight variations in temperature (from 57°C to 64°C) within the interconnected pools led to a marked fluctuation in microbial abundance and diversity. Correlation and PERMANOVA tests provided evidence that temperature was the key environmental factor responsible for microbial community dynamics, while pH, H_(2)S, and SO_2 influenced the abundance of specific microbial groups. When archaeal community composition was analyzed, the majority of detected OTUs correlated negatively with temperature, and few correlated positively with pH. Key Words: Microbial diversity—PhyloChip G3—Acidophilic—Thermophilic—Hot springs—Iceland. Astrobiology 14, xxx–xxx

Agl24 is an ancient archaeal homolog of the eukaryotic N-glycan chitobiose synthesis enzymes

Protein N-glycosylation is a post-translational modification found in organisms of all domains of life. The crenarchaeal N-glycosylation begins with the synthesis of a lipid-linked chitobiose core structure, identical to that in Eukaryotes, although the enzyme catalyzing this reaction remains unknown. Here, we report the identification of a thermostable archaeal β-1,4-N-acetylglucosaminyltransferase, named archaeal glycosylation enzyme 24 (Agl24), responsible for the synthesis of the N-glycan chitobiose core. Biochemical characterization confirmed its function as an inverting β-D-GlcNAc-(1→4)-α-D-GlcNAc-diphosphodolichol glycosyltransferase. Substitution of a conserved histidine residue, found also in the eukaryotic and bacterial homologs, demonstrated its functional importance for Agl24. Furthermore, bioinformatics and structural modeling revealed similarities of Agl24 to the eukaryotic Alg14/13 and a distant relation to the bacterial MurG, which are catalyzing the same or a similar reaction, respectively. Phylogenetic analysis of Alg14/13 homologs indicates that they are ancient in Eukaryotes, either as a lateral transfer or inherited through eukaryogenesis.</p

Functional prediction of proteins from the human gut archaeome

AbstractThe human gastrointestinal tract contains diverse microbial communities, including archaea. Among them,Methanobrevibacter smithiirepresents a highly active and clinically relevant methanogenic archaeon, being involved in gastrointestinal disorders, such as IBD and obesity. Herein, we present an integrated approach using sequence and structure information to improve the annotation ofM. smithiiproteins using advanced protein structure prediction and annotation tools, such as AlphaFold2, trRosetta, ProFunc, and DeepFri. Of an initial set of 873 481 archaeal proteins, we found 707 754 proteins exclusively present in the human gut. Having analysed archaeal proteins together with 87 282 994 bacterial proteins, we identified unique archaeal proteins and archaeal-bacterial homologs. We then predicted and characterized functional domains and structures of 73 unique and homologous archaeal protein clusters linked the human gut andM. smithii. We refined annotations based on the predicted structures, extending existing sequence similarity-based annotations. We identified gut-specific archaeal proteins that may be involved in defense mechanisms, virulence, adhesion, and the degradation of toxic substances. Interestingly, we identified potential glycosyltransferases that could be associated with N-linked and O-glycosylation. Additionally, we found preliminary evidence for interdomain horizontal gene transfer betweenClostridiaspecies andM. smithii, which includessporulation stage V proteins AEand AD. Our study broadens the understanding of archaeal biology, particularlyM. smithii,and highlights the importance of considering both sequence and structure for the prediction of protein function

Functional prediction of proteins from the human gut archaeome

The human gastrointestinal tract contains diverse microbial communities, including archaea. Among them, Methanobrevibacter smithii represents a highly active and clinically relevant methanogenic archaeon, being involved in gastrointestinal disorders, such as inflammatory bowel disease and obesity. Herein, we present an integrated approach using sequence and structure information to improve the annotation of M. smithii proteins using advanced protein structure prediction and annotation tools, such as AlphaFold2, trRosetta, ProFunc, and DeepFri. Of an initial set of 873 481 archaeal proteins, we found 707 754 proteins exclusively present in the human gut. Having analysed archaeal proteins together with 87 282 994 bacterial proteins, we identified unique archaeal proteins and archaeal–bacterial homologs. We then predicted and characterized functional domains and structures of 73 unique and homologous archaeal protein clusters linked the human gut and M. smithii. We refined annotations based on the predicted structures, extending existing sequence similarity-based annotations. We identified gut-specific archaeal proteins that may be involved in defense mechanisms, virulence, adhesion, and the degradation of toxic substances. Interestingly, we identified potential glycosyltransferases that could be associated with N-linked and O-glycosylation. Additionally, we found preliminary evidence for interdomain horizontal gene transfer between Clostridia species and M. smithii, which includes sporulation Stage V proteins AE and AD. Our study broadens the understanding of archaeal biology, particularly M. smithii, and highlights the importance of considering both sequence and structure for the prediction of protein function

Genomic resolution of a cold subsurface aquifer community provides metabolic insights for novel microbes adapted to high CO2 concentrations.

As in many deep underground environments, the microbial communities in subsurface high-CO2 ecosystems remain relatively unexplored. Recent investigations based on single-gene assays revealed a remarkable variety of organisms from little studied phyla in Crystal Geyser (Utah, USA), a site where deeply sourced CO2 -saturated fluids are erupted at the surface. To provide genomic resolution of the metabolisms of these organisms, we used a novel metagenomic approach to recover 227 high-quality genomes from 150 microbial species affiliated with 46 different phylum-level lineages. Bacteria from two novel phylum-level lineages have the capacity for CO2 fixation. Analyses of carbon fixation pathways in all studied organisms revealed that the Wood-Ljungdahl pathway and the Calvin-Benson-Bassham Cycle occurred with the highest frequency, whereas the reverse TCA cycle was little used. We infer that this, and selection for form II RuBisCOs, are adaptions to high CO2 -concentrations. However, many autotrophs can also grow mixotrophically, a strategy that confers metabolic versatility. The assignment of 156 hydrogenases to 90 different organisms suggests that H2 is an important inter-species energy currency even under gaseous CO2 -saturation. Overall, metabolic analyses at the organism level provided insight into the biochemical cycles that support subsurface life under the extreme condition of CO2 saturation

Generator voltage stabilisation for series-hybrid electric vehicles

This paper presents a controller for use in speed control of an internal combustion engine for series-hybrid electric vehicle applications. Particular reference is made to the stability of the rectified DC link voltage under load disturbance. In the system under consideration, the primary power source is a four-cylinder normally aspirated gasoline internal combustion engine, which is mechanically coupled to a three-phase permanent magnet AC generator. The generated AC voltage is subsequently rectified to supply a lead-acid battery, and permanent magnet traction motors via three-phase full bridge power electronic inverters. Two complementary performance objectives exist. Firstly to maintain the internal combustion engine at its optimal operating point, and secondly to supply a stable 42 V supply to the traction drive inverters. Achievement of these goals minimises the transient energy storage requirements at the DC link, with a consequent reduction in both weight and cost. These objectives imply constant velocity operation of the internal combustion engine under external load disturbances and changes in both operating conditions and vehicle speed set-points. An electronically operated throttle allows closed loop engine velocity control. System time delays and nonlinearities render closed loop control design extremely problematic. A model-based controller is designed and shown to be effective in controlling the DC link voltage, resulting in the well-conditioned operation of the hybrid vehicle

SeqCode: a Nomenclatural Code for Prokaryotes described from Sequence Data

Most prokaryotes are not available as pure cultures and therefore ineligible for naming under the rules and recommendations of the International Code of Nomenclature of Prokaryotes (ICNP). Here we summarize the development of the SeqCode, a code of nomenclature under which genome sequences serve as nomenclatural types. This code enables valid publication of names of prokaryotes based upon isolate genome, metagenome-assembled genome or single-amplified genome sequences. Otherwise, it is similar to the ICNP with regard to the formation of names and rules of priority. It operates through the SeqCode Registry (https://seqco.de/), a registration portal through which names and nomenclatural types are registered, validated and linked to metadata. We describe the two paths currently available within SeqCode to register and validate names, including Candidatus names, and provide examples for both. Recommendations on minimal standards for DNA sequences are provided. Thus, the SeqCode provides a reproducible and objective framework for the nomenclature of all prokaryotes regardless of cultivability and facilitates communication across microbiological disciplines