Automated Synthesis of Algal Fucoidan Oligosaccharides.

Fucoidan, a sulfated polysaccharide found in algae, plays a central role in marine carbon sequestration and exhibits a wide array of bioactivities. However, the molecular diversity and structural complexity of fucoidan hinder precise structure-function studies. To address this, we present an automated method for generating well-defined linear and branched α-fucan oligosaccharides. Our syntheses include oligosaccharides with up to 20 cis-glycosidic linkages, diverse branching patterns, and 11 sulfate monoesters. In this study, we demonstrate the utility of these oligosaccharides by (i) characterizing two endo-acting fucoidan glycoside hydrolases (GH107), (ii) utilizing them as standards for NMR studies to confirm suggested structures of algal fucoidans, and (iii) developing a fucoidan microarray. This microarray enabled the screening of the molecular specificity of four monoclonal antibodies (mAb) targeting fucoidan. It was found that mAb BAM4 has cross-reactivity to ß-glucans, while mAb BAM2 has reactivity to fucoidans with 4-O-sulfate esters. Knowledge of the mAb BAM2 epitope specificity provided evidence that a globally abundant marine diatom, Thalassiosira weissflogii, synthesizes a fucoidan with structural homology to those found in brown algae. Automated glycan assembly provides access to fucoidan oligosaccharides. These oligosaccharides provide the basis for molecular level investigations into fucoidan's roles in medicine and carbon sequestration.

Mucus carbohydrate composition correlates with scleractinian coral phylogeny.

The mucus surface layer serves vital functions for scleractinian corals and consists mainly of carbohydrates. Its carbohydrate composition has been suggested to be influenced by environmental conditions (e.g., temperature, nutrients) and microbial pressures (e.g., microbial degradation, microbial coral symbionts), yet to what extend the coral mucus composition is determined by phylogeny remains to be tested. To investigate the variation of mucus carbohydrate compositions among coral species, we analyzed the composition of mucosal carbohydrate building blocks (i.e., monosaccharides) for five species of scleractinian corals, supplemented with previously reported data, to discern overall patterns using cluster analysis. Monosaccharide composition from a total of 23 species (belonging to 14 genera and 11 families) revealed significant differences between two phylogenetic clades that diverged early in the evolutionary history of scleractinian corals (i.e., complex and robust; p = 0.001, R2 = 0.20), mainly driven by the absence of arabinose in the robust clade. Despite considerable differences in environmental conditions and sample analysis protocols applied, coral phylogeny significantly correlated with monosaccharide composition (Mantel test: p < 0.001, R2 = 0.70). These results suggest that coral mucus carbohydrates display phylogenetic dependence and support their essential role in the functioning of corals.

Application of MALDI-MS for characterization of fucoidan hydrolysates and screening of endo-fucoidanase activity.

Brown macroalgae synthesize large amounts of fucoidans, sulfated fucose-containing polysaccharides, in the ocean. Fucoidans are of importance for their recently discovered contribution to marine carbon dioxide sequestration and due to their potential applications in biotechnology and biomedicine. However, fucoidans have high intra- and intermolecular diversity that challenges assignment of structure to biological function and the development of applications. Fucoidan-active enzymes may be used to simplify this diversity by producing defined oligosaccharides more applicable for structural refinement, characterization, and structure to function assignment for example via bioassays. In this study, we combined MALDI mass spectrometry with biocatalysis to show that the endo-fucoidanases P5AFcnA and Wv323 can produce defined oligosaccharide structures directly from unrefined macroalgal biomass. P5AFcnA released oligosaccharides from seven commercial fucoidan extracts in addition to unrefined biomass of three macroalgae species indicating a broadly applicable approach reproducible across 10 species. Both MALDI-TOF/TOF and AP-MALDI-Orbitrap systems were used, demonstrating that the approach is not instrument-specific and exploiting their combined high-throughput and high-resolution capabilities. Overall, the combination of MALDI-MS and endo-fucoidanase assays offers high-throughput evaluation of fucoidan samples and also enables extraction of defined oligosaccharides of known structure from unrefined seaweed biomass.

Taxonomic and functional stability overrules seasonality in polar benthic microbiomes.

Coastal shelf sediments are hot spots of organic matter mineralization. They receive up to 50% of primary production, which, in higher latitudes, is strongly seasonal. Polar and temperate benthic bacterial communities, however, show a stable composition based on comparative 16S rRNA gene sequencing despite different microbial activity levels. Here, we aimed to resolve this contradiction by identifying seasonal changes at the functional level, in particular with respect to algal polysaccharide degradation genes, by combining metagenomics, metatranscriptomics, and glycan analysis in sandy surface sediments from Isfjorden, Svalbard. Gene expressions of diverse carbohydrate-active enzymes changed between winter and spring. For example, ß-1,3-glucosidases (e.g. GH30, GH17, GH16) degrading laminarin, an energy storage molecule of algae, were elevated in spring, while enzymes related to α-glucan degradation were expressed in both seasons with maxima in winter (e.g. GH63, GH13_18, and GH15). Also, the expression of GH23 involved in peptidoglycan degradation was prevalent, which is in line with recycling of bacterial biomass. Sugar extractions from bulk sediments were low in concentrations during winter but higher in spring samples, with glucose constituting the largest fraction of measured monosaccharides (84% ± 14%). In porewater, glycan concentrations were ~18-fold higher than in overlying seawater (1107 ± 484 vs. 62 ± 101 µg C l-1) and were depleted in glucose. Our data indicate that microbial communities in sandy sediments digest and transform labile parts of photosynthesis-derived particulate organic matter and likely release more stable, glucose-depleted residual glycans of unknown structures, quantities, and residence times into the ocean, thus modulating the glycan composition of marine coastal waters.

Low-level resource partitioning supports coexistence among functionally redundant bacteria during successional dynamics.

Members of microbial communities can substantially overlap in substrate use. However, what enables functionally redundant microorganisms to coassemble or even stably coexist remains poorly understood. Here, we show that during unstable successional dynamics on complex, natural organic matter, functionally redundant bacteria can coexist by partitioning low-concentration substrates even though they compete for one simple, dominant substrate. We allowed ocean microbial communities to self-assemble on leachates of the brown seaweed Fucus vesiculosus and then analyzed the competition among 10 taxonomically diverse isolates representing two distinct stages of the succession. All, but two isolates, exhibited an average of 90% ± 6% pairwise overlap in resource use, and functional redundancy of isolates from the same assembly stage was higher than that from between assembly stages, leading us to construct a simpler four-isolate community with two isolates from each of the early and late stages. We found that, although the short-term dynamics of the four-isolate communities in F. vesiculosus leachate was dependent on initial isolate ratios, in the long term, the four isolates stably coexist in F. vesiculosus leachate, albeit with some strains at low abundance. We therefore explored the potential for nonredundant substrate use by genomic content analysis and RNA expression patterns. This analysis revealed that the four isolates mainly differed in peripheral metabolic pathways, such as the ability to degrade pyrimidine, leucine, and tyrosine, as well as aromatic substrates. These results highlight the importance of fine-scale differences in metabolic strategies for supporting the frequently observed coexistence of large numbers of rare organisms in natural microbiomes.

Alpha-glucans from bacterial necromass indicate an intra-population loop within the marine carbon cycle.

Phytoplankton blooms provoke bacterioplankton blooms, from which bacterial biomass (necromass) is released via increased zooplankton grazing and viral lysis. While bacterial consumption of algal biomass during blooms is well-studied, little is known about the concurrent recycling of these substantial amounts of bacterial necromass. We demonstrate that bacterial biomass, such as bacterial alpha-glucan storage polysaccharides, generated from the consumption of algal organic matter, is reused and thus itself a major bacterial carbon source in vitro and during a diatom-dominated bloom. We highlight conserved enzymes and binding proteins of dominant bloom-responder clades that are presumably involved in the recycling of bacterial alpha-glucan by members of the bacterial community. We furthermore demonstrate that the corresponding protein machineries can be specifically induced by extracted alpha-glucan-rich bacterial polysaccharide extracts. This recycling of bacterial necromass likely constitutes a large-scale intra-population energy conservation mechanism that keeps substantial amounts of carbon in a dedicated part of the microbial loop.