Höfundur: Kristín Edda Gylfadóttir

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Enzymatic depolymerization of alginate by two novel thermostable alginate lyases from Rhodothermus marinus

Tengiliður

Ólafur H. Friðjónsson

Fagstjóri

olafur@matis.is

Alginate (alginic acid) is a linear polysaccharide, wherein (1→4)-linked β-D-mannuronic acid and its C5 epimer, α-L-guluronic acid, are arranged in varying sequences. Alginate lyases catalyze the depolymerization of alginate, thereby cleaving the (1→4) glycosidic linkages between the monomers by a β-elimination mechanism, to yield unsaturated 4-deoxy-L–erythro-hex-4-enopyranosyluronic acid (Δ) at the non-reducing end of resulting oligosaccharides (α-L–erythro configuration) or, depending on the enzyme, the unsaturated monosaccharide itself. In solution, the released free unsaturated monomer product is further hydrated in a spontaneous (keto-enol tautomerization) process to form two cyclic stereoisomers. In this study, two alginate lyase genes, designated alyRm3 and alyRm4, from the marine thermophilic bacterium Rhodothermus marinus (strain MAT378), were cloned and expressed in Escherichia coli. The recombinant enzymes were characterized, and their substrate specificity and product structures determined. AlyRm3 (PL39) and AlyRm4 (PL17) are among the most thermophilic and thermostable alginate lyases described to date with temperature optimum of activity at ∼75 and 81°C, respectively. The pH optimum of activity of AlyRm3 is ∼5.5 and AlyRm4 at pH 6.5. Detailed NMR analysis of the incubation products demonstrated that AlyRm3 is an endolytic lyase, while AlyRm4 is an exolytic lyase, cleaving monomers from the non-reducing end of oligo/poly-alginates.

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Molecular Characterization of a DNA Polymerase from Thermus thermophilus MAT72 Phage vB_Tt72: A Novel Type-A Family Enzyme with Strong Proofreading Activity

Tengiliður

Ólafur H. Friðjónsson

Fagstjóri

olafur@matis.is

We present a structural and functional analysis of the DNA polymerase of thermophilic Thermus thermophilus MAT72 phage vB_Tt72. The enzyme shows low sequence identity (<30%) to the members of the type-A family of DNA polymerases, except for two yet uncharacterized DNA polymerases of T. thermophilus phages: φYS40 (91%) and φTMA (90%). The Tt72 polA gene does not complement the Escherichia coli polA− mutant in replicating polA-dependent plasmid replicons. It encodes a 703-aa protein with a predicted molecular weight of 80,490 and an isoelectric point of 5.49. The enzyme contains a nucleotidyltransferase domain and a 3′-5′ exonuclease domain that is engaged in proofreading. Recombinant enzyme with His-tag at the N-terminus was overproduced in E. coli, subsequently purified by immobilized metal affinity chromatography, and biochemically characterized. The enzyme exists in solution in monomeric form and shows optimum activity at pH 8.5, 25 mM KCl, and 0.5 mM Mg2+. Site-directed analysis proved that highly-conserved residues D15, E17, D78, D180, and D184 in 3′-5′ exonuclease and D384 and D615 in the nucleotidyltransferase domain are critical for the enzyme’s activity. Despite the source of origin, the Tt72 DNA polymerase has not proven to be highly thermoresistant, with a temperature optimum at 55 °C. Above 60 °C, the rapid loss of function follows with no activity > 75 °C. However, during heat treatment (10 min at 75 °C), trehalose, trimethylamine N-oxide, and betaine protected the enzyme against thermal inactivation. A midpoint of thermal denaturation at Tm = 74.6 °C (ΔHcal = 2.05 × 104 cal mol−1) and circular dichroism spectra > 60 °C indicate the enzyme’s moderate thermal stability.

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Two Fish in a Pod. Mislabelling on Board Threatens Sustainability in Mixed Fisheries  

Tengiliður

Jónas Rúnar Viðarsson

Áherslusviðsstjóri

jonas@matis.is

Accuracy in reporting captures is a key element to achieve fisheries sustainability. However, identification of the catches might be a challenge when two or more species are morphologically similar and caught jointly, like the mixed fisheries of black hakes in East Atlantic African waters. Black hakes (Merluccius senegalensis and M. polli) are tough to differentiate without previous training due to their high morphological resemblance. The two species are managed as a single stock, although the biological differences between them suggest the need of a separate management. In this study, a total of 806 black hakes were visually identified by fishers on deck of fishing vessels operating in Mauritania and Senegal waters, then assigned to a species by sequencing 450bp of the Mitochondrial Control Region. Comparing the results with visual identification we found 31.4% of the total catch were incorrectly labelled on board by the fishermen. The accuracy of the fishers’ identification depended on the depth of capture and on fish size, larger individuals caught from deeper waters being more correctly assigned to M. polli. Mislabelling biased to M. polli suggests that M. senegalensis, already catalogued as endangered, is being underreported, which could endanger the conservation of this species and threaten the sustainability of black hake fisheries. Our results highlight the need for separate evaluation of the stocks in mixed fisheries for morphologically similar fish. Thus, monitoring through DNA barcoding in the very first step of the seafood chain surveys would improve accurate species delimitation and reduce its impact on the correct assessment of the stocks.

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HOMEFOOD randomized trial-beneficial effects of 6-month nutrition therapy on body weight and physical function in older adults at risk for malnutrition after hospital discharge

Tengiliður

Kolbrún Sveinsdóttir

Verkefnastjóri

kolbrun.sveinsdottir@matis.is

Background/objectives: Malnutrition is common among older adults. Dietary intervention studies in older adults aiming to improve anthropometrics measures and physical function have been inconsistent. We aimed to investigate the effects of nutrition therapy in combination with home delivered meals and oral nutritional supplements (ONS) in community-dwelling older adults discharged from hospital.

Methods: A total of 106 participants (>65 years) were randomized into the intervention group (n = 53) and into the control group (n = 53). The intervention group received individual nutrition therapy (five in person visits and three phone calls) and freely delivered energy- and protein- rich foods, while the control group received standard care. Dietary intake, anthropometrics, and short physical performance battery (SPPB) were assessed at baseline and at endpoint.

Results: Energy intake at baseline was similar in both groups (~1500 kcal at the hospital) but there was a significant increase in energy intake and body weight in the intervention group (+919 kcal/day and 1.7 kg, P < 0.001 in both cases) during the study period, compared to a significant decrease in both measures among controls (-815 kcal/day and -3.5 kg, P < 0.001 in both cases). SPPB score increased significantly in the intervention group while no changes were observed among controls.

Conclusions: Most Icelandic older adults experience substantial weight loss after hospital discharge when receiving current standard care. However, a 6-month multi-component nutrition therapy, provided by a clinical nutritionist in combination with freely delivered supplemental energy- and protein-dense foods has beneficial effects on body weight, physical function, and nutritional status.

Study registration: This study was registered at ClinicalTrials.gov ( NCT03995303 ).

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Basalt-Hosted Microbial Communities in the Subsurface of the Young Volcanic Island of Surtsey, Iceland

Tengiliður

Pauline Vannier

Verkefnastjóri

pauline.vannier@matis.is

The island of Surtsey was formed in 1963–1967 on the offshore Icelandic volcanic rift zone. It offers a unique opportunity to study the subsurface biosphere in newly formed oceanic crust and an associated hydrothermal-seawater system, whose maximum temperature is currently above 120°C at about 100m below surface. Here, we present new insights into the diversity, distribution, and abundance of microorganisms in the subsurface of the island, 50years after its creation. Samples, including basaltic tuff drill cores and associated fluids acquired at successive depths as well as surface fumes from fumaroles, were collected during expedition 5059 of the International Continental Scientific Drilling Program specifically designed to collect microbiological samples. Results of this microbial survey are investigated with 16S rRNA gene amplicon sequencing and scanning electron microscopy. To distinguish endemic microbial taxa of subsurface rocks from potential contaminants present in the drilling fluid, we use both methodological and computational strategies. Our 16S rRNA gene analysis results expose diverse and distinct microbial communities in the drill cores and the borehole fluid samples, which harbor thermophiles in high abundance. Whereas some taxonomic lineages detected across these habitats remain uncharacterized (e.g., Acetothermiia, Ammonifexales), our results highlight potential residents of the subsurface that could be identified at lower taxonomic rank such as Thermaerobacter, BRH-c8a (Desulfallas-Sporotomaculum), Thioalkalimicrobium, and Sulfurospirillum. Microscopy images reveal possible biotic structures attached to the basaltic substrate. Finally, microbial colonization of the newly formed basaltic crust and the metabolic potential are discussed on the basis of the data.

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Rhodothermus bifroesti sp. nov., a thermophilic bacterium isolated from the basaltic subsurface of the volcanic island Surtsey

Tengiliður

Pauline Vannier

Verkefnastjóri

pauline.vannier@matis.is

Novel thermophilic heterotrophic bacteria were isolated from the subsurface of the volcanic island Surtsey off the south coast of Iceland. The strains were isolated from tephra core and borehole fluid samples collected below 70 m depth. The Gram-negative bacteria were rod-shaped (0.3-0.4 µm wide, 1.5-7 µm long), aerobic, non-sporulating and non-motile. Optimal growth was observed at 70 °C, at pH 7-7.5 and with 1% NaCl. Phylogenetic analysis identified the strains as members of the genus Rhodothermus. The type strain, ISCAR-7401T, was genetically distinct from its closest relatives Rhodothermus marinus DSM 4252T and Rhodothermus profundi PRI 2902T based on 16S rRNA gene sequence similarity (95.81 and 96.01%, respectively), genomic average nucleotide identity (73.73 and 72.61%, respectively) and digital DNA-DNA hybridization (17.6 and 16.9%, respectively). The major fatty acids of ISCAR-7401T were iso-C17:0, anteiso-C15:0, anteiso-C17:0 and iso-C15:0 (>10 %). The major isoprenoid quinone was MK-7 while phosphatidylethanolamine, diphosphatidylglycerol, an unidentified aminophospholipid and a phospholipid were the predominant polar lipid components. Based on comparative chemotaxonomic, genomic and phylogenetic analyses, we propose that the isolated strain represents a novel species of the genus Rhodothermus with the name Rhodothermus bifroesti sp. nov. The type strain is ISCAR-7401T(=DSM 112103T=CIP 111906T).

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Results-based management in practice: Lessons learnt and policy recommendations from the implementation of RBM in European fisheries outside Europe

Tengiliður

Jónas Rúnar Viðarsson

Áherslusviðsstjóri

jonas@matis.is

This paper summarizes the lessons learnt from the implementation of a practical approach to Results-based Management (RBM) within the EU H2020 FarFish project, where RBM is conceptualized as a contract situation. From these lessons a set of policy recommendations for the improvement of the fisheries management through the adoption of RBM for EU vessels fishing in international- and Sustainable FisheriesPartnership Agreements (SFPA) waters were put forward. The policy recommendations are based on key project outputs and additional semi-structured interviews on the experiences of project partners. This exercise revealed advancements and shortcomings for the broader implementation of a more participatory, inclusive, and responsive approach to fisheries governance. Meaningful and effective participation was a pivotal factor for the success of the process. Absence of relevant bodies, sectors and fleet segments proved detrimental throughout all cases. The importance of scoping, goal setting and timing of the actions, as well as managing realistic expectations within the RBM, were highlighted. Data availability and accountability was pivotal, as in cases were cooperation succeeded, data was made available, and knowledge was expanded and enriched. The implementation of this approach to RBM showed great potential when extended to other aspects of fisheries’ management, fishing categories and fleets. This structured approach to RBM provides a promising alternative to current fisheries management systems across the world.

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Crystal structure of DNA polymerase I from Thermus phage G20c

Tengiliður

Guðmundur Óli Hreggviðsson

Stefnumótandi sérfræðingur

gudmundo@matis.is

This study describes the structure of DNA polymerase I from Thermus phage G20c, termed PolI_G20c. This is the first structure of a DNA polymerase originating from a group of related thermophilic bacteriophages infecting Thermus thermophilus, including phages G20c, TSP4, P74-26, P23-45 and phiFA and the novel phage Tth15-6. Sequence and structural analysis of PolI_G20c revealed a 3′–5′ exonuclease domain and a DNA polymerase domain, and activity screening confirmed that both domains were functional. No functional 5′–3′ exonuclease domain was present. Structural analysis also revealed a novel specific structure motif, here termed SβαR, that was not previously identified in any polymerase belonging to the DNA polymerases I (or the DNA polymerase A family). The SβαR motif did not show any homology to the sequences or structures of known DNA polymerases. The exception was the sequence conservation of the residues in this motif in putative DNA polymerases encoded in the genomes of a group of thermophilic phages related to Thermus phage G20c. The structure of PolI_G20c was determined with the aid of another structure that was determined in parallel and was used as a model for molecular replacement. This other structure was of a 3′–5′ exonuclease termed ExnV1. The cloned and expressed gene encoding ExnV1 was isolated from a thermophilic virus metagenome that was collected from several hot springs in Iceland. The structure of ExnV1, which contains the novel SβαR motif, was first determined to 2.19 Å resolution. With these data at hand, the structure of PolI_G20c was determined to 2.97 Å resolution. The structures of PolI_G20c and ExnV1 are most similar to those of the Klenow fragment of DNA polymerase I (PDB entry 2kzz) from Escherichia coli, DNA polymerase I from Geobacillus stearo­thermophilus (PDB entry 1knc) and Taq polymerase (PDB entry 1bgx) from Thermus aquaticus.

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Crystal structure and initial characterization of a novel archaeal-like Holliday junction-resolving enzyme from Thermus thermophilus phage Tth15-6

Tengiliður

Ólafur H. Friðjónsson

Fagstjóri

olafur@matis.is

This study describes the production, characterization and structure determination of a novel Holliday junction-resolving enzyme. The enzyme, termed Hjc_15-6, is encoded in the genome of phage Tth15-6, which infects Thermus thermophilus. Hjc_15-6 was heterologously produced in Escherichia coli and high yields of soluble and biologically active recombinant enzyme were obtained in both complex and defined media. Amino-acid sequence and structure comparison suggested that the enzyme belongs to a group of enzymes classified as archaeal Holliday junction-resolving enzymes, which are typically divalent metal ion-binding dimers that are able to cleave X-shaped dsDNA-Holliday junctions (Hjs). The crystal structure of Hjc_15-6 was determined to 2.5 Å resolution using the selenomethionine single-wavelength anomalous dispersion method. To our knowledge, this is the first crystal structure of an Hj-resolving enzyme originating from a bacteriophage that can be classified as an archaeal type of Hj-resolving enzyme. As such, it represents a new fold for Hj-resolving enzymes from phages. Characterization of the structure of Hjc_15-6 suggests that it may form a dimer, or even a homodimer of dimers, and activity studies show endonuclease activity towards Hjs. Furthermore, based on sequence analysis it is proposed that Hjc_15-6 has a three-part catalytic motif corresponding to E-SD-EVK, and this motif may be common among other Hj-resolving enzymes originating from thermophilic bacteriophages.

Skýrslur

Phosphorus uptake and requirement from feed and water by Atlantic salmon (Salmo salar, Linnaeus, 1758) juveniles in freshwater Recirculating Aquaculture System

Útgefið:

20/07/2023

Höfundar:

Helda Kizhakkuden Sajeev, David Sutter, Wolfgang Koppe, Sven-Ole Meiske & Georges Lamborelle

Styrkt af:

Mowi feed AS

Tengiliður

Georges Lamborelle

Stöðvarstjóri tilraunaeldisstöðvar

georges@matis.is

Þessi skýrsla er lokuð / This report is closed

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