Bacterial Epibiotic Communities of Ubiquitous and Abundant Marine Diatoms Are Distinct in Short- and Long-Term Associations

Interactions between phytoplankton and bacteria play a central role in mediating biogeochemical cycling and food web structure in the ocean. The cosmopolitan diatoms Thalassiosira and Chaetoceros often dominate phytoplankton communities in marine systems. Past studies of diatom-bacterial associations have employed community-level methods and culture-based or natural diatom populations. Although bacterial assemblages attached to individual diatoms represents tight associations little is known on their makeup or interactions. Here, we examined the epibiotic bacteria of 436 Thalassiosira and 329 Chaetoceros single cells isolated from natural samples and collection cultures, regarded here as short- and long-term associations, respectively. Epibiotic microbiota of single diatom hosts was analyzed by cultivation and by cloning-sequencing of 16S rRNA genes obtained from whole-genome amplification products. The prevalence of epibiotic bacteria was higher in cultures and dependent of the host species. Culture approaches demonstrated that both diatoms carry distinct bacterial communities in short- and long-term associations. Bacterial epibonts, commonly associated with phytoplankton, were repeatedly isolated from cells of diatom collection cultures but were not recovered from environmental cells. Our results suggest that in controlled laboratory culture conditions bacterial–diatom and bacterial–bacterial interactions select for a simplified, but specific, epibiotic microbiota shaped and adapted for long-term associations

Dissection of cell wall assembly dynamics during early embryogenesis in the brown alga Fucus

Zygotes from Fucus species have been used extensively to study cell polarization and rhizoid outgrowth, and in this model system cell wall deposition aligns with the establishment of polarity. Monoclonal antibodies are essential tools for the in situ analysis of cell wall glycans, and here we report the characteristics of six monoclonal antibodies to alginates (BAM6–BAM11). The use of these, in conjunction with monoclonal antibodies to brown algal sulfated fucans, has enabled the study of the developmental dynamics of the Fucus zygote cell walls. Young zygotes are spherical and all alginate epitopes are deposited uniformly following cellulose deposition. At germination, sulfated fucans are secreted in the growing rhizoid wall. The redistribution of cell wall epitopes was investigated during treatments that cause reorientation of the growth axis (change in light direction) or disrupt rhizoid development (arabinogalactan-protein-reactive Yariv reagent). Alginate modeling was drastically impaired in the latter, and both treatments cause a redistribution of highly sulfated fucan epitopes. The dynamics of cell wall glycans in this system have been visualized in situ for the first time, leading to an enhanced understanding of the early developmental mechanisms of Fucus species. These sets of monoclonal antibodies significantly extend the available molecular tools for brown algal cell wall studies

Insoluble (1 → 3), (1 → 4)-β-D-glucan is a component of cell walls in brown algae (Phaeophyceae) and is masked by alginates in tissues

International audienceBrown algae are photosynthetic multicellular marine organisms. They belong to the phylum of Stramenopiles, which are not closely related to land plants and green algae. Brown algae share common evolutionary features with other photosynthetic and multicellular organisms, including a carbohydrate-rich cell-wall. Brown algal cell walls are composed predominantly of the polyanionic polysaccharides alginates and fucose-containing sulfated polysaccharides. These polymers are prevalent over neutral and crystalline components, which are believed to be mostly, if not exclusively, cellulose. In an attempt to better understand brown algal cell walls, we performed an extensive glycan array analysis of a wide range of brown algal species. Here we provide the first demonstration that mixed-linkage (1 → 3), (1 → 4)-β-d-glucan (MLG) is common in brown algal cell walls. Ultra-Performance Liquid Chromatography analyses indicate that MLG in brown algae solely consists of trisaccharide units of contiguous (1 → 4)-β-linked glucose residues joined by (1 → 3)-β-linkages. This regular conformation may allow long stretches of the molecule to align and to form well-structured microfibrils. At the tissue level, immunofluorescence studies indicate that MLG epitopes in brown algae are unmasked by a pre-treatment with alginate lyases to remove alginates. These findings are further discussed in terms of the origin and evolution of MLG in the Stramenopile lineage

Genetic analyses unravel the crucial role of a horizontally acquired alginate lyase for brown algal biomass degradation by Z obellia galactanivorans

International audienceComprehension of the degradation of macroalgal polysaccharides suffers from the lack of genetic tools for model marine bacteria, despite their importance for coastal ecosystem functions. We developed such tools for Zobellia galactanivorans, an algae-associated flavobacterium that digests many polysaccharides, including alginate. These tools were used to investigate the biological role of AlyA1, the only Z. galactanivorans alginate lyase known to be secreted in soluble form and to have a recognizable carbohydrate-binding domain. A deletion mutant, ΔalyA1, grew as well as the wild type on soluble alginate but was deficient in soluble secreted alginate lyase activity and in digestion of and growth on alginate gels and algal tissues. Thus, AlyA1 appears to be essential for optimal attack of alginate in intact cell walls. alyA1 appears to have been recently acquired via horizontal transfer from marine Actinobacteria, conferring an adaptive advantage that might benefit other algae-associated bacteria by exposing new substrate niches. The genetic tools described here function in diverse members of the phylum Bacteroidetes and should facilitate analyses of polysaccharide degradation systems and many other processes in these common but understudied bacteria

Double blind microarray-based polysaccharide profiling enables parallel identification of uncharacterized polysaccharides and carbohydrate-binding proteins with unknown specificities

Abstract Marine algae are one of the largest sources of carbon on the planet. The microbial degradation of algal polysaccharides to their constitutive sugars is a cornerstone in the global carbon cycle in oceans. Marine polysaccharides are highly complex and heterogeneous, and poorly understood. This is also true for marine microbial proteins that specifically degrade these substrates and when characterized, they are frequently ascribed to new protein families. Marine (meta)genomic datasets contain large numbers of genes with functions putatively assigned to carbohydrate processing, but for which empirical biochemical activity is lacking. There is a paucity of knowledge on both sides of this protein/carbohydrate relationship. Addressing this ‘double blind’ problem requires high throughput strategies that allow large scale screening of protein activities, and polysaccharide occurrence. Glycan microarrays, in particular the Comprehensive Microarray Polymer Profiling (CoMPP) method, are powerful in screening large collections of glycans and we described the integration of this technology to a medium throughput protein expression system focused on marine genes. This methodology (Double Blind CoMPP or DB-CoMPP) enables us to characterize novel polysaccharide-binding proteins and to relate their ligands to algal clades. This data further indicate the potential of the DB-CoMPP technique to accommodate samples of all biological sources