the genome of Rhodothermus marinus DSM 4253 encodes six glycoside hydrolases (GH) classified under GH family 3 (GH3): RmBgl3A, RmBgl3B, RmBgl3C, RmXyl3A, RmXyl3B and RmNag3. The biochemical function, modelled 3D-structure, gene cluster and evolutionary relationships of each of these enzymes were studied. The six enzymes were clustered into three major evolutionary lineages of GH3: β-N-acetyl-glucosaminidases, β-1,4-glucosidases/β-xylosidases and macrolide β-glucosidases. the RmNag3 with additional β-lactamase domain clustered with the deepest rooted GH3-lineage of β-N-acetyl-glucosaminidases and was active on acetyl-chitooligosaccharides. RmBgl3B displayed β-1,4-glucosidase activity and was the only representative of the lineage clustered with macrolide β-glucosidases from Actinomycetes. the β-xylosidases, RmXyl3A and RmXyl3B, and the β-glucosidases RmBgl3A and RmBgl3C clustered within the major β-glucosidases/β-xylosidases evolutionary lineage. RmXyl3A and RmXyl3B showed β-xylosidase activity with different specificities for para-nitrophenyl (pNP)-linked substrates and xylooligosaccharides. RmBgl3A displayed β-1,4-glucosidase/β-xylosidase activity while RmBgl3C was active on pnp-β-Glc and β-1,3-1,4-linked glucosyl disaccharides. Putative polysaccharide utilization gene clusters were also investigated for both R. marinus DSM 4253 and DSM 4252 t (homolog strain). The analysis showed that in the homolog strain DSM 4252 t Rmar_1080 (RmXyl3A) and Rmar_1081 (RmXyl3B) are parts of a putative polysaccharide utilization locus (PUL) for xylan utilization. Marine extremophilic biotopes, such as hot springs and hydrothermal vents, harbour diverse microbes hitherto underexploited and unexplored. Recent genomic studies show that many of the species, especially those found in coastal geothermal areas surrounded by profusion of carbohydrate rich biomass (seaweeds as well as terrestrial species), contain a wide array of novel glycoside hydrolases (GHs) 1,2. Thermostable GHs have numerous applications in different fields, making marine thermophiles targets for prospecting of industrially interesting enzymes 3,4. Rhodothermus marinus are Gram-negative marine thermophilic bacteria, previously classified under the phylum Bacteroidetes, but recently assigned to the new phylum Rhodothermaeota 5. The type-species was isolated from a coastal hot spring on the NorthWest coast of Iceland and has an optimum temperature of 65 °C and is slightly halophilic 6. R. marinus can utilize a variety of sugars as carbon sources and produces a wide range of GHs 6-12. Sequence analysis shows that the R. marinus genome contains a large number of genes encoding GH enzymes, many of which are secreted extracellularly; yet, several of them appear to be attached to the cell surface 13. These putative enzymes include six members of GH family 3 (GH3) (RmBgl3A, RmBgl3B, RmBgl3C, RmXyl3A, RmXyl3B and RmNag3), which have not yet been studied.
The Virus-X—Viral Metagenomics for Innovation Value—project was a scientific expedition to explore and exploit uncharted territory of genetic diversity in extreme natural environments such as geothermal hot springs and deep-sea ocean ecosystems. Specifically, the project was set to analyse and exploit viral metagenomes with the ultimate goal of developing new gene products with high innovation value for applications in biotechnology, pharmaceutical, medical, and the life science sectors. Viral gene pool analysis is also essential to obtain fundamental insight into ecosystem dynamics and to investigate how viruses influence the evolution of microbes and multicellular organisms. The Virus-X Consortium, established in 2016, included experts from eight European countries. The unique approach based on high throughput bioinformatics technologies combined with structural and functional studies resulted in the development of a biodiscovery pipeline of significant capacity and scale. The activities within the Virus-X consortium cover the entire range from bioprospecting and methods development in bioinformatics to protein production and characterisation, with the final goal of translating our results into new products for the bioeconomy. The significant impact the consortium made in all of these areas was possible due to the successful cooperation between expert teams that worked together to solve a complex scientific problem using state-of-the-art technologies as well as developing novel tools to explore the virosphere, widely considered as the last great frontier of life.
The lytic cassette proteins XepA and YomS from Bacillus subtilis prophages have been characterized and it was found that only XepA establishes cytotoxic activity in plaque assays. The crystal structures of both proteins show a unique pentameric assembly, in which YomS adopts a very similar fold to the C-terminal domain of the XepA dumbbell pentamer. The overall architecture of XepA, with the N-terminal domain subunits resembling cytoplasmic membrane-binding C2-domain folds, suggests that any lytic functionality could be based on disruption of the proton motive force of the cytoplasmic membrane, which induces cell lysis.
While small organic molecules generally crystallize forming tightly packed lattices with little solvent content, proteins form air‐sensitive high‐solvent‐content crystals. Here, the crystallization and full structure analysis of a novel recombinant 10 kDa protein corresponding to the C‐terminal domain of a putative U32 peptidase are reported. The orthorhombic crystal contained only 24.5% solvent and is therefore among the most tightly packed protein lattices ever reported.
Prevotella copri DSM18205 T is a human gut bacterium, suggested as a next‐generation probiotic. To utilize it as such, it is, however, necessary to grow the species in a reproducible manner. Prevotella copri has previously been reported to be highly sensitive to oxygen, and hence difficult to isolate and cultivate. This study presents successful batch cultivation strategies for viable strain inoculations and growth in both serum bottles and a stirred tank bioreactor (STR), without the use of an anaerobic chamber, as long as the cells were kept in the exponential growth phase. A low headspace volume in the STR was important to reach high cell density. P . copri utilized xylose cultivated in Peptone Yeast Xylose medium (PYX medium), resulting in a comparable growth rate and metabolite production as in Peptone Yeast Glucose medium (PYG medium) in batch cultivations at pH 7.2.Up to 5 g/L of the carbon source was consumed, leading to the production of succinic acid, acetic acid, and formic acid, and cell densities (OD 620 nm ) in the range 6−7.5. The highest yield of produced succinic acid was 0.63 ± 0.05 g/g glucose in PYG medium cultivations and 0.88 ± 0.06 g/g xylose in PYX medium cultivations.
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