The Structure of RdDddP from Roseobacter denitrificans Reveals That DMSP Lyases in the DddP-Family Are Metalloenzymes

Marine microbes degrade dimethylsulfoniopropionate (DMSP), which is produced in large quantities by marine algae and plants, with DMSP lyases into acrylate and the gas dimethyl sulfide (DMS). Approximately 10% of the DMS vents from the sea into the atmosphere and this emission returns sulfur, which arrives in the sea through rivers and runoff, back to terrestrial systems via clouds and rain. Despite their key role in this sulfur cycle DMSP lyases are poorly understood at the molecular level. Here we report the first X-ray crystal structure of the putative DMSP lyase RdDddP from Roseobacter denitrificans, which belongs to the abundant DddP family. This structure, determined to 2.15 Å resolution, shows that RdDddP is a homodimeric metalloprotein with a binuclear center of two metal ions located 2.7 Å apart in the active site of the enzyme. Consistent with the crystallographic data, inductively coupled plasma mass spectrometry (ICP-MS) and total reflection X-ray fluorescence (TRXF) revealed the bound metal species to be primarily iron. A 3D structure guided analysis of environmental DddP lyase sequences elucidated the critical residues for metal binding are invariant, suggesting all proteins in the DddP family are metalloenzymes

Re-hierarquização e Extrapolações para o Limite do Conjunto de Base Completo.

Um método sugerido previamente para calcular a energia de correlação no limite do conjunto de base completo pela redesignação dos números hierárquicos, e o uso do esquema de extrapolação unified singlet- and triplet-pair é aplicado a um conjunto de prova de 106 sistemas. A aproximação é utilizada para obter os valores extrapolados para energia de correlação, energia de atomização, anisotropia e polarizabilidade média no limite do conjunto de base completo, através de teoria de perturbação de segunda ordem de Møller-Plesset, método de coupled-cluster com excitações simples e duplas e coupled-cluster com excitações simples e duplas com correções triplas perturbativas. Uma boa concordância com as melhores estimativas disponíveis é obtida, mesmo quando o par de números hierárquicos (d, t) é usado para realizar a extrapolação. Com isso, é concebível justificar que não há razão física forte para excluir as energias dulpa-zeta em extrapolações, especialmente se a base é calibrada para obedecer ao modelo teórico. Além disso, um esquema simples de extrapolação unificado de um parâmetro é sugerido para extrapolar a energia de correlação de valência para o conjunto de base completo em espécies formadas por átomos de H até Ne. A performance do novo modelo é avaliada para a energia de correlação com um conjunto de de dados de 106 sistemas e, para polarizabilidade média, em um conjunto de 8 moléculas. Para as energias de correlação, os resultados são excelentes, na maioria das vezes melhores do que quando extrapolado com os mais populares protocolos de dois parâmetros disponíveis na literatura. Para a polarizabilidade, os resultados mostram uma melhora em relação aos valores ab initio, e uma boa concordância com os dados experimentais

Single cell fluorescence imaging of glycan uptake by intestinal bacteria

Microbes in the intestines of mammals degrade dietary glycans for energy and growth. The pathways required for polysaccharide utilization are functionally diverse; moreover, they are unequally dispersed between bacterial genomes. Hence, assigning metabolic phenotypes to genotypes remains a challenge in microbiome research. Here we demonstrate that glycan uptake in gut bacteria can be visualized with fluorescent glycan conjugates (FGCs) using epifluorescence microscopy. Yeast α-mannan and rhamnogalacturonan-II, two structurally distinct glycans from the cell walls of yeast and plants, respectively, were fluorescently labeled and fed to Bacteroides thetaiotaomicron VPI-5482. Wild-type cells rapidly consumed the FGCs and became fluorescent; whereas, strains that had deleted pathways for glycan degradation and transport were non-fluorescent. Uptake of FGCs, therefore, is direct evidence of genetic function and provides a direct method to assess specific glycan metabolism in intestinal bacteria at the single cell level.</p

Fucoid brown algae inject fucoidan carbon into the ocean

Peer reviewe

A marine bacterial enzymatic cascade degrades the algal polysaccharide ulvan

International audienc

Analyse structurale et fonctionnelle des enzymes du système agarolytique de la flavobactérie marine zobellia galactanivorans

Zobellia galactanivorans is a marine Flavobacterie capable of degrading complex polysaccharides such as agar widely used in biotechnology and agribusiness. In the marine ecosystem photosynthetic organisms such as algae and cyanobacteria are the main producers of organic carbon. This source of carbon is used by marine bacteria that secrete glycoside hydrolases to attack algae or hydrolyze the walls of decaying algae. To understand the mechanisms of carbon recycling from these preferential nutrition areas we decided to analyze the system agarolytique Z. galactanivorans. The complete bacterial genome revealed the presence of nine agarases GH family 16, confirming that Z. galactanivorans has a complex system agarolytique to degrade the natural resource of agar. Seven original sequences were overexpressed through a medium-speed cloning strategy. A new beta-agarase (AGAD) was purified to homogeneity, and then crystallized and diffraction data were collected at a resolution of 1.5 Å. The structure was solved by molecular replacement and biochemical characterization was performed. In addition, the first two? -porphyranases PorA and PorB were identified, crystallized and characterized biochemically. The diversity of agarolytiques enzymes reflects the biological adaptations shown by certain bacteria to accommodate the variability agarose wall of red algae.Zobellia galactanivorans est une flavobactérie marine capable de dégrader des polysaccharides complexes tels que l’agar qui est largement utilisé en biotechnologie et agroalimentaire. Dans l’écosystème marin les organismes photosynthétiques tels que les algues et les cyanobactéries sont les principaux producteurs de carbone organique. Cette source de carbone est utilisée par les bactéries marines qui sécrètent des glycosides hydrolases afin d’attaquer les algues ou d’hydrolyser la paroi des algues en décomposition. Pour comprendre les mécanismes de recyclage du carbone à partir de ces zones de nutrition préférentielles nous avons décidé d’analyser le système agarolytique de Z. galactanivorans. Le génome bactérien complet a mis en évidence la présence de neuf agarases de la famille GH 16, confirmant que Z. galactanivorans possède un système agarolytique complexe pour dégrader cette ressource naturel qu’est l’agar. Sept séquences originales ont été surexprimées grâce à une stratégie de clonage à moyen débit. Une nouvelle ß-agarase (AgaD) a été purifiée à l’homogénéité, puis cristallisée et les données de diffraction ont été collectées à une résolution de 1.5 Å. La structure a été résolue par remplacement moléculaire et la caractérisation biochimique a été réalisée. De plus, les deux premières ?-porphyranases PorA et PorB ont été identifiées, cristallisées et caractérisées biochimiquement. La diversité d’enzymes agarolytiques reflète les adaptations biologiques dont ont fait preuve certaines bactéries pour s’accommoder à la grande variabilité de l’agarose de la paroi des algues rouges

Polysaccharide structures in HMWDOM and POM at the North Sea detected by carbohydrate microarray analysis

Here, we traced the abundance of 27 polysaccharide epitopes in dissolved and particulate organic matter along a three month diatom bloom period in the North Sea. We used a bioanalytic approach based on carbohydrate microarrays and monoclonal antibodies (mAbs). Details describing the data: Carbohydrate microarray data show the relative polysaccharide abundance (antibody signal intensity) detected in samples harvested during a spring phytoplankton bloom period (21 sampling dates) in the North Sea (54˚11.3'N, 7˚54.0'E). Samples include high molecular weight dissolved organic matter (HMWDOM) and particulate organic matter (POM). Polysaccharides from all samples were sequentially extracted with the solvents H2O, 50 mM EDTA pH 7.5 and 4 M NaOH with 0.1% w/v NaBH4. A library of identical microarrays was created, each populated with the same time-series of extracted polysaccharides. These microarrays were then individually incubated with polysaccharide-specific mAbs or carbohydrate binding modules (CBMs) as probes, which specifically bind to a single polysaccharide epitope. The binding of probes to polysaccharide epitopes on the microarrays was detected using a secondary antibody coupled to alkaline phosphatase, which converts its substrate into a coloured product, the amount of which correlates with polysaccharide concentration. Antibody signal intensity was quantified and the highest signal value in the data set for HMWDOM and for POM was set to 100 and all other values were normalised accordingly. The temporal dynamics but not the absolute number should be compared between HMWDOM and POM pools as they required independent normalisation since their sampling was different. A cut-off of 5 arbitrary units was applied and all probe profiles where in at least one date an antibody positive signal (value ≥ 5) was detected are included in the data set. The epitope recognised by each probe is shown at the top of each column and the name of the corresponding mAb or CBM is depicted in parentheses. Size fractions correspond to: POM 10 µm, over 10 µm; POM 3 µm, between 10 and 3 µm; POM 0.2 µm, between 3 and 0.2 µm; HMWDOM, between 0.2 µm and 1 kDa. HG, homogalacturonan; DE, degree of esterification; AGP, arabinogalactan protein; GlcA, glucuronic acid. This microarray data set reveals the temporal dynamics of 27 polysaccharide epitopes in HMWDOM and POM

A marine bacterial enzymatic cascade degrades the algal polysaccharide ulvan

Marine seaweeds increasingly grow into extensive algal blooms, which are detrimental to coastal ecosystems, tourism and aquaculture. However, algal biomass is also emerging as a sustainable raw material for the bioeconomy. The potential exploitation of algae is hindered by our limited knowledge of the microbial pathways—and hence the distinct biochemical functions of the enzymes involved—that convert algal polysaccharides into oligo- and monosaccharides. Understanding these processes would be essential, however, for applications such as the fermentation of algal biomass into bioethanol or other value-added compounds. Here, we describe the metabolic pathway that enables the marine flavobacterium Formosa agariphila to degrade ulvan, the main cell wall polysaccharide of bloom-forming Ulva species. The pathway involves 12 biochemically characterized carbohydrate-active enzymes, including two polysaccharide lyases, three sulfatases and seven glycoside hydrolases that sequentially break down ulvan into fermentable monosaccharides. This way, the enzymes turn a previously unexploited renewable into a valuable and ecologically sustainable bioresource

Monosaccharide composition in HMWDOM during a microalgae bloom period in the North Sea

We analysed the monosaccharide composition in high molecular weight dissolved organic matter (HMWDOM) during a three month diatom bloom period in the North Sea (54˚11.3'N, 7˚54.0'E). HMWDOM samples were concentrated by tangential flow filtration (TFF). These were hydrolysed with acid into monomers and quantified by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Details describing the data: A total of 19 samples were analysed including two technical replicates. Each sample corresponds to 100 L of 0.2 μm-filtered seawater that were concentrated to a final volume of 0.5 L by TFF with 1 kDa cassettes. Therefore samples contain the size fraction between 0.2 µm and 1 kDa. The TFF-concentrated HMWDOM samples were analysed and data show their monosaccharide composition as mean relative abundance (in molarity)

Polysaccharides present in HMWDOM and POM during a microalgae bloom period in the North Sea

High molecular weight dissolved organic matter (HMWDOM) and particulate organic matter (POM) samples were harvested during a spring phytoplankton bloom period in the North Sea for about three months. Polysaccharides from all HMWDOM and POM samples were extracted and analysed by carbohydrate microarray analysis. Additionally, glycans in the HMWDOM samples were also studied by monosaccharide analysis