Marine microalgae (phytoplankton) mediate almost half of the worldwide photosynthetic carbon dioxide fixation and therefore play a pivotal role in global carbon cycling, most prominently during massive phytoplankton blooms. Phytoplankton biomass consists of considerable proportions of polysaccharides, substantial parts of which are rapidly remineralized by heterotrophic bacteria. We analyzed the diversity, activity, and functional potential of such polysaccharide-degrading bacteria in different size fractions during a diverse spring phytoplankton bloom at Helgoland Roads (southern North Sea) at high temporal resolution using microscopic, physicochemical, biodiversity, metagenome, and metaproteome analyses. Prominent active 0.2–3 µm free-living clades comprised Aurantivirga, “Formosa”, Cd. Prosiliicoccus, NS4, NS5, Amylibacter, Planktomarina, SAR11 Ia, SAR92, and SAR86, whereas BD1-7, Stappiaceae, Nitrincolaceae, Methylophagaceae, Sulfitobacter, NS9, Polaribacter, Lentimonas, CL500-3, Algibacter, and Glaciecola dominated 3–10 µm and > 10 µm particles. Particle-attached bacteria were more diverse and exhibited more dynamic adaptive shifts over time in terms of taxonomic composition and repertoires of encoded polysaccharide-targeting enzymes. In total, 305 species-level metagenome-assembled genomes were obtained, including 152 particle-attached bacteria, 100 of which were novel for the sampling site with 76 representing new species. Compared to free-living bacteria, they featured on average larger metagenome-assembled genomes with higher proportions of polysaccharide utilization loci. The latter were predicted to target a broader spectrum of polysaccharide substrates, ranging from readily soluble, simple structured storage polysaccharides (e.g., laminarin, α-glucans) to less soluble, complex structural, or secreted polysaccharides (e.g., xylans, cellulose, pectins). In particular, the potential to target poorly soluble or complex polysaccharides was more widespread among abundant and active particle-attached bacteria. Particle-attached bacteria represented only 1

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. Phytoplankton blooms provoke bacterioplankton blooms, from which bacterial biomass (necromass) is released via zooplankton grazing and viral lysis. Here, Beidler et al. show that the bacterial biomass, including alpha-glucan polysaccharides generated from the consumption of algal organic matter, is reused by microbes in vitro and during a diatom-dominated bloom.

Laminarin, a β(1,3)-glucan, serves as a storage polysaccharide in marine microalgae such as diatoms. Its abundance, water solubility and simple structure make it an appealing substrate for marine bacteria. Consequently, many marine bacteria have evolved strategies to scavenge and decompose laminarin, employing carbohydrate-binding modules (CBMs) as crucial components. In this study, we characterized two previously unassigned domains as laminarin-binding CBMs in multimodular proteins from the marine bacterium Christiangramia forsetii KT0803 T , thereby introducing the new laminarin-binding CBM families CBM102 and CBM103. We identified four CBM102s in a surface glycan-binding protein (SGBP) and a single CBM103 linked to a glycoside hydrolase module from family 16 (GH16_3). Our analysis revealed that both modular proteins have an elongated shape, with GH16_3 exhibiting greater flexibility than SGBP. This flexibility may aid in the recognition and/or degradation of laminarin, while the constraints in SGBP could facilitate the docking of laminarin onto the bacterial surface. Exploration of bacterial metagenome-assembled genomes (MAGs) from phytoplankton blooms in the North Sea showed that both laminarin-binding CBM families are widespread among marine Bacteroidota . The high protein abundance of CBM102- and CBM103-containing proteins during phytoplankton blooms further emphasizes their significance in marine laminarin utilization.

SusC/D-like proteins are essential components of glycan utilization machineries in Bacteroidota , but remain unknown in other bacterial phyla. The glycan-binding SusD-like protein forms a ‘lid’ on top of the SusC-like TonB-dependent transporter (TBDT) and both are structurally designed to function as a complex in sugar uptake. In comparison, Gammaproteobacteria import glycans using ‘classical’ TBDTs without an accessory SusD-like protein. We have now identified a SusD-like protein and a SusC-like TBDT in a fructan polysaccharide utilization locus (PUL) of the marine gammaproteobacterium Pseudoalteromonas distincta , which are tandemly organized as in Bacteroidota . Proteome analysis revealed an increased production of PUL-encoded proteins during growth on inulin- and levan-type fructans. However, P. distincta preferred inulin over plant-derived levan and hardly grew on bacterial levan. Further analysis showed that the SusD-like protein has a weak affinity (Ka 43 M-1) for oligosaccharides from inulin. The PUL-encoded glycoside hydrolase from family 32 (GH32) hydrolyzes inulin and plant-derived levan, but also has a low activity on bacterial levan, which confirms the growth experiments. Comparative genomics identified further SusC/D-like proteins in Gammaproteobacteria genomes, most of which (83%) were encoded in fructan PULs. Significance statement It is a mystery why the Gram-negative Bacteroidota and Gammaproteobacteria use different transport systems for glycan utilization. In contrast to this observation, we found that the gammaproteobacterium Pseudoalteromonas distincta uses a Bacteroidota -like transport complex in fructan uptake, composed of a glycan-binding SusD-like protein and a SusC-like TonB-dependent transporter. Proteome analysis revealed that the SusC/D-like proteins and a fructan-degrading enzyme are produced at high levels in fructan-grown cells. Weak affinity of the SusD-like protein to inulin oligosaccharides and enzyme activity on fructans confirmed their fructan specificity. Further SusC/D-like pairs were detected in other Gammaproteobacteria , mostly associated with fructan-related gene clusters. This study thus describes marine fructan use by Gammaproteobacteria and indicates an exchange of protein functions between phyla. ### Competing Interest Statement The authors have declared no competing interest.

Blooming microalgae (phytoplankton) release diverse organic molecules that fuel the marine pools of dissolved and particulate organic matter. A highly specialized community of heterotrophic bacteria rapidly remineralizes substantial parts of this organic matter in the sun-lit upper ocean. In particular, microalgae produce large quantities of various organosulfur compounds that can serve as carbon and sulfur sources for bacteria. Here, we report on the analyses of a time series of previously generated 30 long-read metagenomes, 30 corresponding deeply sequenced short-read metatranscriptomes and 15 metaproteomes from 0.2-3 μm size fractions that we sampled in 2020 during a biphasic phytoplankton bloom in the German Bight (Southern North Sea). We analyzed the assembled contigs as well as 70 bacterial metagenome-assembled genomes that recruited the highest transcript numbers with respect to the utilization of methyl sulfur compounds (dimethylsulfoniopropionate (DMSP), dimethyl sulfide (DMS), dimethyl sulfone (DMSO2)), C3-sulfonates (2,3-dihydroxypropane-1-sulfonate (DHPS), 3-sulfolactate, 3-sulfopyruvate) and 2-aminoethanesulfonic acid (taurine). We observed a pronounced resource partitioning among bacterial clades that utilize distinct organosulfur compounds, which may explain successions of these clades during the studied bloom. Alphaproteobacteria were the most active and degraded a variety of organosulfonates via various metabolic routes. However, we also found previously underreported roles of members of the Bacteroidota and Gammaproteobacteria as efficient degraders of DMSP, DMS, and DMSO2. One striking observation was a strong preference for DMSP cleavage in Bacteroidota as opposed to DMSP demethylation in Alphaproteobacteria and indications for a particular proficiency for taurine utilization in Ilumatobacter_A and Acidimicrobiia .

The ß-(1,3)-glucan laminarin functions as storage polysaccharide in marine stramenophiles such as diatoms. Laminarin is abundant, water-soluble and structured simply, making it an attractive substrate for marine bacteria. As a consequence, many marine bacteria have developed competitive strategies to scavenge and decompose laminarin, which involves carbohydrate-binding modules (CBMs) as key players. We therefore functionally and structurally characterized two yet unassigned domains as laminarin-binding CBMs in multimodular proteins from our model bacterium Christiangramia forsetii KT0803 T , hereby unveiling the novel laminarin-binding CBM families CBMxx and CBMyy (official CAZy numbering will be provided upon acceptance of the manuscript in a peer-reviewed journal). We discovered four CBMxx repeats in a surface glycan-binding protein (SGBP) and a single CBMyy combined with a glycoside hydrolase module from family 16 (GH16_3). Our analyses revealed that both modular proteins have an elongated shape, and that the GH16_3 displayed a higher flexibility than the SGBP. While motility of both polypeptide chains may facilitate recognition and/or degradation of laminarin, constraints in the SGBP may support docking of laminarin onto the bacterial surface. The exploration of bacterial metagenome-assembled genomes (MAGs) from phytoplankton blooms in the North Sea revealed that both laminarin-binding CBM families are widely distributed among marine Bacteroidota , illustrating the high adaptability of modularity in sugar-binding and -degrading proteins. High expression of CBMxx- and CBMyy-containing proteins during phytoplankton blooms further underpins their importance in marine laminarin usage.

Marine Bacteroidetes that degrade polysaccharides contribute to carbon cycling in the ocean. Organic matter, including glycans from terrestrial plants, might enter the oceans through rivers. Whether marine bacteria degrade structurally related glycans from diverse sources including terrestrial plants and marine algae was previously unknown. We show that the marine bacterium Flavimarina sp. Hel_I_48 encodes two polysaccharide utilization loci (PULs) which degrade xylans from terrestrial plants and marine algae. Biochemical experiments revealed activity and specificity of the encoded xylanases and associated enzymes of these PULs. Proteomics indicated that these genomic regions respond to glucuronoxylans and arabinoxylans. Substrate specificities of key enzymes suggest dedicated metabolic pathways for xylan utilization. Some of the xylanases were active on different xylans with the conserved β-1,4-linked xylose main chain. Enzyme activity was consistent with growth curves showing Flavimarina sp. Hel_I_48 uses structurally different xylans. The observed abundance of related xylan-degrading enzyme repertoires in genomes of other marine Bacteroidetes indicates similar activities are common in the ocean. The here presented data show that certain marine bacteria are genetically and biochemically variable enough to access parts of structurally diverse xylans from terrestrial plants as well as from marine algal sources.

The polysaccharide β-mannan, which is common in terrestrial plants but unknown in microalgae, was recently detected during diatom blooms. We identified a β-mannan polysaccharide utilization locus (PUL) in the genome of the marine flavobacterium Muricauda sp. MAR_2010_75. Proteomics showed β-mannan induced translation of 22 proteins encoded within the PUL. Biochemical and structural analyses deduced the enzymatic cascade for β-mannan utilization. A conserved GH26 β-mannanase with endo-activity depolymerized the β-mannan. Consistent with the biochemistry, X-ray crystallography showed the typical TIM-barrel fold of related enzymes found in terrestrial β-mannan degraders. Structural and biochemical analyses of a second GH26 allowed the prediction of an exo-activity on shorter manno-gluco oligosaccharides. Further analysis demonstrated exo-α-1,6-galactosidase- and endo-β-1,4-glucanase activity of the PUL-encoded GH27 and GH5_26, respectively, indicating the target substrate is a galactoglucomannan. Epitope deletion assays with mannanases as analytic tools indicate the presence of β-mannan in the diatoms Coscinodiscus wailesii and Chaetoceros affinis. Mannanases from the PUL were active on diatom β-mannan and polysaccharide extracts sampled during a microalgal bloom at the North Sea. Together these results demonstrate that marine microorganisms use a conserved enzymatic cascade to degrade β-mannans of marine and terrestrial origin and that this metabolic pathway plays a role in marine carbon cycling.

Formaldehyde is a toxic metabolite that is formed in large quantities during bacterial utilization of the methoxy sugar 6‐ O ‐methyl‐ d ‐galactose, an abundant monosaccharide in the red algal polysaccharide porphyran. Marine bacteria capable of metabolizing porphyran must therefore possess suitable detoxification systems for formaldehyde. We demonstrate here that detoxification of formaldehyde in the marine Flavobacterium Zobellia galactanivorans proceeds via the ribulose monophosphate pathway. Simultaneously, we show that the genes encoding the key enzymes of this pathway are important for maintaining high formaldehyde resistance. Additionally, these genes are upregulated in the presence of porphyran, allowing us to connect porphyran degradation to the detoxification of formed formaldehyde.

Microplastics in marine ecosystems are colonized by diverse prokaryotic and eukaryotic communities. How these communities and their functional profiles are shaped by the artificial surfaces remains broadly unknown. In order to close this knowledge gap, we set up an in situ experiment with pellets of the polyolefin polymer polyethylene (PE), the aromatic hydrocarbon polymer polystyrene (PS), and wooden beads along a coastal to estuarine gradient in the Baltic Sea, Germany. We used an integrated metagenomics/metaproteomics approach to evaluate the genomic potential as well as protein expression levels of aquatic plastic biofilms. Our results suggest that material properties had a minor influence on the plastic-associated assemblages, as genomic and proteomic profiles of communities associated with the structurally different polymers PE and PS were highly similar, hence polymer-unspecific. Instead, it seemed that these communities were shaped by biogeographic factors. Wood, on the other hand, induced the formation of substrate-specific biofilms and served as nutrient source itself. Our study indicates that, while PE and PS microplastics may be relevant in the photic zone as opportunistic colonization grounds for phototrophic microorganisms, they appear not to be subject to biodegradation or serve as vectors for pathogenic microorganisms in marine habitats.

Marine algae catalyze half of all global photosynthetic production of carbohydrates. Owing to their fast growth rates, Ulva spp. rapidly produce substantial amounts of carbohydrate-rich biomass and represent an emerging renewable energy and carbon resource. Their major cell wall polysaccharide is the anionic carbohydrate ulvan. Here, we describe a new enzymatic degradation pathway of the marine bacterium Formosa agariphila for ulvan oligosaccharides involving unsaturated uronic acid at the nonreducing end linked to rhamnose-3-sulfate and glucuronic or iduronic acid (Δ-Rha3S-GlcA/IdoA-Rha3S). Notably, we discovered a new dehydratase (P29_PDnc) acting on the nonreducing end of ulvan oligosaccharides, i.e., GlcA/IdoA-Rha3S, forming the aforementioned unsaturated uronic acid residue. This residue represents the substrate for GH105 glycoside hydrolases, which complements the enzymatic degradation pathway including one ulvan lyase, one multimodular sulfatase, three glycoside hydrolases, and the dehydratase P29_PDnc, the latter being described for the first time. Our research thus shows that the oligosaccharide dehydratase is involved in the degradation of carboxylated polysaccharides into monosaccharides.

Outer membrane extensions are common in many marine bacteria. However, the function of these surface enlargements or extracellular compartments is poorly understood. Using a combined approach of microscopy and subproteome analyses, we therefore examined Pseudoalteromonas distincta ANT/505, an Antarctic polysaccharide degrading gamma-proteobacterium. P. distincta produced outer membrane vesicles (MV) and vesicle chains (VC) on polysaccharide and non-polysaccharide carbon sources during the exponential and stationary growth phase. Surface structures of carbohydrate-grown cells were equipped with increased levels of highly substrate-specific proteins. At the same time, proteins encoded in all other polysaccharide degradation-related genomic regions were also detected in MV and VC samples under all growth conditions, indicating a basal expression. In addition, two alkaline phosphatases were highly abundant under non-limiting phosphate conditions. Surface structures may thus allow rapid sensing and fast responses in nutritionally deprived environments. It may also facilitate efficient carbohydrate processing and reduce loss of substrates and enzymes by diffusion as important adaptions to the aquatic ecosystem.

The formation of sinking particles in the ocean, which promote carbon sequestration into deeper water and sediments, involves algal polysaccharides acting as an adhesive, binding together molecules, cells and minerals. These as yet unidentified adhesive polysaccharides must resist degradation by bacterial enzymes or else they dissolve and particles disassemble before exporting carbon. Here, using monoclonal antibodies as analytical tools, we trace the abundance of 27 polysaccharide epitopes in dissolved and particulate organic matter during a series of diatom blooms in the North Sea, and discover a fucose-containing sulphated polysaccharide (FCSP) that resists enzymatic degradation, accumulates and aggregates. Previously only known as a macroalgal polysaccharide, we find FCSP to be secreted by several globally abundant diatom species including the genera Chaetoceros and Thalassiosira. These findings provide evidence for a novel polysaccharide candidate to contribute to carbon sequestration in the ocean.

Algal blooms produce large quantities of organic matter that is subsequently remineralised by bacterial heterotrophs. Polysaccharide is a primary component of algal biomass. It has been hypothesised that individual bacterial heterotrophic niches during algal blooms are in part determined by the available polysaccharide substrates present. Measurement of the expression of TonB-dependent transporters, often specific for polysaccharide uptake, might serve as a proxy for assessing bacterial polysaccharide consumption over time. To investigate this, we present here high-resolution metaproteomic and metagenomic datasets from bacterioplankton of the 2016 spring phytoplankton bloom at Helgoland island in the southern North Sea, and expression profiles of TonB-dependent transporters during the bloom, which demonstrate the importance of both the Gammaproteobacteria and the Bacteroidetes as degraders of algal polysaccharide. TonB-dependent transporters were the most highly expressed protein class, split approximately evenly between the Gammaproteobacteria and Bacteroidetes, and totalling on average 16.7% of all detected proteins during the bloom. About 93% of these were predicted to take up organic matter, and for about 12% of the TonB-dependent transporters, we predicted a specific target polysaccharide class. Most significantly, we observed a change in substrate specificities of the expressed transporters over time, which was not reflected in the corresponding metagenomic data. From this, we conclude that algal cell wall-related compounds containing fucose, mannose, and xylose were mostly utilised in later bloom stages, whereas glucose-based algal and bacterial storage molecules including laminarin, glycogen, and starch were used throughout. Quantification of transporters could therefore be key for understanding marine carbon cycling.