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Carbohydrates are chemically and structurally diverse, represent a substantial fraction of marine organic matter and are key substrates for heterotrophic microbes. Studies on carbohydrate utilisation by marine microbes have been centred on phytoplankton blooms in temperate regions, while far less is known from high-latitude waters and during later seasonal stages. Here, we combine glycan microarrays and analytical chromatography with metagenomics and metatranscriptomics to show the spatial heterogeneity in glycan distribution and their utilisation by microbes in Atlantic waters of the Arctic during late summer. The composition and abundance of monomers and glycan structures in POM varied with location and depth. Complex fucose-containing sulfated polysaccharides, known to accumulate in the ocean, were consistently detected, suggesting limited degradation by microbes. In contrast, the more labile β-1,3-glucan exhibited a patchy distribution, indicating local variations in primary productivity and rapid utilisation. Metatranscriptomics showed active and dynamic microbial populations that targeted specific glycans. Gene transcription of carbohydrate-active enzymes revealed narrow substrate niches for specialists, involving compounds such as α-mannans and alginate, along with the targeting of communal substrates, such as laminarin, by multiple populations. The observed spatial heterogeneity indicates that local biological and physical processes continue to shape the carbohydrate pool during late summer in high latitude waters and microbial populations are active and responsive to such changes.

It is generally recognized that phages are a mortality factor for their bacterial hosts. This could be particularly true in spring phytoplankton blooms, which are known to be closely followed by a highly specialized bacterial community. We hypothesized that phages modulate these dense heterotrophic bacteria successions following phytoplankton blooms. In this study, we focused on Flavobacteriia, because they are main responders during these blooms and have an important role in the degradation of polysaccharides. A cultivation-based approach was used, obtaining 44 lytic flavobacterial phages (flavophages), representing twelve new species from two viral realms. Taxonomic analysis allowed us to delineate ten new phage genera and ten new families, from which nine and four, respectively, had no previously cultivated representatives. Genomic analysis predicted various life styles and genomic replication strategies. A likely eukaryote-associated host habitat was reflected in the gene content of some of the flavophages. Detection in cellular metagenomes and by direct-plating showed that part of these phages were actively replicating in the environment during the 2018 spring bloom. Furthermore, CRISPR/Cas spacers and re-isolation during two consecutive years suggested that, at least part of the new flavophages are stable components of the microbial community in the North Sea. Together, our results indicate that these diverse flavophages have the potential to modulate their respective host populations.

Blooms of marine microalgae play a pivotal role in global carbon cycling. Such blooms entail successive blooms of specialized clades of planktonic bacteria that remineralize algal biomass. We investigated the bacterioplankton response to a bloom in the German Bight in spring 2020. Metagenome sequencing at 30 time-points allowed reconstruction of 251 metagenome-assembled genomes (MAGs), 245 representing as yet uncultured species, while corresponding metatranscriptome sequencing highlighted 50 particularly active MAGs. Together with algae, copepod, protist and bacteria diversity and abundance data in combination with physico-chemical data and antibody-based saccharide measurements, we demonstrate (i) how dissolved primary photoassimilated algal and secondary bacterial storage glycans shape the bacterioplankton community composition, and (ii) how grazing on higher trophic levels determines the release of these abundant glycans. We thus elucidate principles governing how bacterioplankton clades respond to algal blooms and collectively remineralize gigatons of carbon annually on a global scale.

The Arctic Ocean is experiencing unprecedented changes as a result of climate warming, necessitating detailed analyses on the ecology and dynamics of biological communities to understand current and future ecosystem shifts. Here we show the pronounced impact that variations in Atlantic water influx and sea-ice cover have on bacterial communities in the East Greenland Current (Fram Strait) using two, 2-year high-resolution amplicon datasets and an annual cycle of long-read metagenomes. Densely ice-covered polar waters harboured a temporally stable, resident microbiome. In contrast, low-ice cover and Atlantic water influx shifted community dominance to seasonally fluctuating populations enriched in genes for phytoplankton-derived organic matter degradation. We identified signature populations associated with distinct oceanographic conditions and predicted their ecological niches. Our study indicates progressing “Biological Atlantification” in the Arctic Ocean, where the niche space of Arctic bacterial populations will diminish, while communities that taxonomically and functionally resemble those in temperate oceans will become more widespread.

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., The fate of ocean carbon is determined by the balance between primary productivity and heterotrophic breakdown of that photosynthate. Here the authors show that diatoms produce a polysaccharide that resists bacterial degradation, accumulates, aggregates and stores carbon during spring blooms.

Background Metagenomics and single cell genomics provide a window into the genetic repertoire of yet uncultivated microorganisms, but both methods are usually taxonomically untargeted. The combination of fluorescence in situ hybridization (FISH) and fluorescence activated cell sorting (FACS) has the potential to enrich taxonomically well-defined clades for genomic analyses. Methods Cells hybridized with a taxon-specific FISH probe are enriched based on their fluorescence signal via flow cytometric cell sorting. A recently developed FISH procedure, the hybridization chain reaction (HCR)-FISH, provides the high signal intensities required for flow cytometric sorting while maintaining the integrity of the cellular DNA for subsequent genome sequencing. Sorted cells are subjected to shotgun sequencing, resulting in targeted metagenomes of low diversity. Results Pure cultures of different taxonomic groups were used to (1) adapt and optimize the HCR-FISH protocol and (2) assess the effects of various cell fixation methods on both the signal intensity for cell sorting and the quality of subsequent genome amplification and sequencing. Best results were obtained for ethanol-fixed cells in terms of both HCR-FISH signal intensity and genome assembly quality. Our newly developed pipeline was successfully applied to a marine plankton sample from the North Sea yielding good quality metagenome assembled genomes from a yet uncultivated flavobacterial clade. Conclusions With the developed pipeline, targeted metagenomes at various taxonomic levels can be efficiently retrieved from environmental samples. The resulting metagenome assembled genomes allow for the description of yet uncharacterized microbial clades.

Marine heterotrophic bacteria contribute considerably to global carbon cycling, in part by utilizing phytoplankton-derived polysaccharides. The patterns and rates of two different polysaccharide utilization modes - extracellular hydrolysis and selfish uptake - have previously been found to change during spring phytoplankton bloom events. Here we investigated seasonal changes in bacterial utilization of three polysaccharides, laminarin, xylan and chondroitin sulfate. Strong seasonal differences were apparent in mode and speed of polysaccharide utilization, as well as in bacterial community compositions. Compared to the winter month of February, during the spring bloom in May, polysaccharide utilization was detected earlier in the incubations and a higher portion of all bacteria took up laminarin selfishly. Highest polysaccharide utilization was measured in June and September, mediated by bacterial communities that were significantly different from spring assemblages. Extensive selfish laminarin uptake, for example, was detectible within a few hours in June, while extracellular hydrolysis of chondroitin was dominant in September. In addition to the well-known Bacteroidota and Gammaproteobacteria clades, the numerically minor verrucomicrobial clade Pedosphaeraceae could be identified as a rapid laminarin utilizer. In summary, polysaccharide utilization proved highly variable over the seasons, both in mode and speed, and also by the bacterial clades involved.

Gene clusters rich in carbohydrate-active enzymes within Flavobacteriia genera provide a competitiveness for their hosts to degrade diatom-derived polysaccharides. One such widely distributed polysaccharide is glucuronomannan, a main cell wall component of diatoms. A conserved gene cluster putatively degrading glucuronomannan was found previously among various flavobacterial taxa in marine metagenomes. Here, we aimed to visualize two glycoside hydrolase family 92 genes coding for α-mannosidases with fluorescently-labeled polynucleotide probes using direct-geneFISH. Reliable in situ localization of single-copy genes was achieved with an efficiency up to 74% not only in the flavobacterial strains Polaribacter Hel1_33_49 and Formosa Hel1_33_131 but also in planktonic samples from the North Sea. In combination with high-resolution microscopy, direct-geneFISH gave visual evidence of the contrasting lifestyles of closely related Polaribacter species in those samples and allowed for the determination of gene distribution among attached and free-living cells. We also detected highly similar GH92 genes in yet unidentified taxa by broadening probe specificities, enabling a visualization of the functional trait in subpopulations across the borders of species and genera. Such a quantitative insight into the niche separation of flavobacterial taxa complements our understanding of the ecology of polysaccharide-degrading bacteria beyond omics-based techniques on a single-cell level.

Winogradskyella is a genus within the phylum Bacteroidetes with a clear marine origin. Most members of this genus have been found associated with marine animals and algae, but also with inorganic surfaces such as sand. In this study, we analyzed genomes of eleven species recently isolated from surface seawater samples from the North Sea during a single spring algae bloom. Corresponding metagenomes yielded a single Candidatus species for this genus. All species in culture, with the exception of W. ursingii, affiliated with a Winogradskyella lineage characterized by large genomes (~4.3 ± 0.4 Mb), with high complexity in their carbohydrate and protein degradation genes. Specifically, the polysaccharide utilization loci (PULs) were diverse within each individual strain, indicating large substrate versatility. Although present in the North Sea, the abundances of these strains were at, or below, the detection limit of the metagenomes. In contrast, the single species, classified as Candidatus W. atlantica, to which all North Sea MAGs belonged, affiliated with a lineage in which the cultivated representatives showed small genomes of ~3.0–3.5 Mb, with the MAGs having ~2.3 Mb. In Ca. W. atlantica, genome streamlining has apparently resulted in the loss of biosynthesis pathways for several amino acids including arginine, methionine, leucine and valine, and the PUL loci were reduced to a single one for utilizing laminarin. This as-yet uncultivated species seems to capitalize on sporadically abundant substrates that are released by algae blooms, mainly laminarin. We also suggest that this streamlined genome might be responsible for the lack of growth on plates for this Candidatus species, in contrast to growth of the less abundant but coexisting members of the genus.

Marine algae annually sequester petagrams of carbon dioxide into polysaccharides, which are a central metabolic fuel for marine carbon cycling. Diatom microalgae produce sulfated polysaccharides containing methyl pentoses that are challenging to degrade for bacteria compared to other monomers, implicating these sugars as a potential carbon sink. Free-living bacteria occurring in phytoplankton blooms that specialise on consuming microalgal sugars, containing fucose and rhamnose remain unknown. Here, genomic and proteomic data indicate that small, coccoid, free-living Verrucomicrobiota specialise in fucose and rhamnose consumption during spring algal blooms in the North Sea. Verrucomicrobiota cell abundance was coupled with the algae bloom onset and accounted for up to 8% of the bacterioplankton. Glycoside hydrolases, sulfatases, and bacterial microcompartments, critical proteins for the consumption of fucosylated and sulfated polysaccharides, were actively expressed during consecutive spring bloom events. These specialised pathways were assigned to novel and discrete candidate species of the Akkermansiaceae and Puniceicoccaceae families, which we here describe as Candidatus Mariakkermansia forsetii and Candidatus Fucivorax forsetii. Moreover, our results suggest specialised metabolic pathways could determine the fate of complex polysaccharides consumed during algae blooms. Thus the sequestration of phytoplankton organic matter via methyl pentose sugars likely depend on the activity of specialised Verrucomicrobiota populations.

One of the most abundant sources of organic carbon in the ocean is glycolate, the secretion of which by marine phytoplankton results in an estimated annual flux of one petagram of glycolate in marine environments1. Although it is generally accepted that glycolate is oxidized to glyoxylate by marine bacteria2–4, the further fate of this C2 metabolite is not well understood. Here we show that ubiquitous marine Proteobacteria are able to assimilate glyoxylate via the β-hydroxyaspartate cycle (BHAC) that was originally proposed 56 years ago5. We elucidate the biochemistry of the BHAC and describe the structure of its key enzymes, including a previously unknown primary imine reductase. Overall, the BHAC enables the direct production of oxaloacetate from glyoxylate through only four enzymatic steps, representing—to our knowledge—the most efficient glyoxylate assimilation route described to date. Analysis of marine metagenomes shows that the BHAC is globally distributed and on average 20-fold more abundant than the glycerate pathway, the only other known pathway for net glyoxylate assimilation. In a field study of a phytoplankton bloom, we show that glycolate is present in high nanomolar concentrations and taken up by prokaryotes at rates that allow a full turnover of the glycolate pool within one week. During the bloom, genes that encode BHAC key enzymes are present in up to 1.5% of the bacterial community and actively transcribed, supporting the role of the BHAC in glycolate assimilation and suggesting a previously undescribed trophic interaction between autotrophic phytoplankton and heterotrophic bacterioplankton. Marine Proteobacteria use the β-hydroxyaspartate cycle to assimilate glycolate, which is secreted by algae on a petagram scale, providing evidence of a previously undescribed trophic interaction between autotrophic phytoplankton and heterotrophic bacterioplankton.

Since the discovery of archaeoplankton in 1992, the euryarchaeotal Marine Group II (MGII) remains uncultured and less understood than other planktonic archaea. We characterized the seasonal dynamics of MGII populations in the southern North Sea on a genomic and microscopic level over the course of four years. We recovered 34 metagenome-assembled genomes (MAGs) of MGIIa and MGIIb that corroborated proteorhodopsin-based photoheterotrophic lifestyles. However, MGIIa and MGIIb MAG genome sizes differed considerably (~1.9 vs. ~1.4 Mbp), as did their transporter, peptidase, flagella and sulfate assimilation gene repertoires. MGIIb populations were characteristic of winter samples, whereas MGIIa accounted for up to 23% of the community at the beginning of summer. Both clades consisted of annually recurring, sequence-discrete populations with low intra-population sequence diversity. Oligotyping of filtered cell-size fractions and microscopy consistently suggested that MGII cells were predominantly free-living. Cells were coccoid and ~0.7 µm in diameter, likely resulting in grazing avoidance. Based on multiple lines of evidence, we propose distinct niche adaptations of MGIIa and MGIIb Euryarchaeota populations that are characteristic of summer and winter conditions in the coastal North Sea.

Niche concept is a core tenet of ecology that has recently been applied in marine microbial research to describe the partitioning of taxa based either on adaptations to specific conditions across environments or on adaptations to specialised substrates. In this study, we combine spatiotemporal dynamics and predicted substrate utilisation to describe species-level niche partitioning within the NS5 Marine Group. Despite NS5 representing one of the most abundant marine flavobacterial clades from across the world’s oceans, our knowledge on their phylogenetic diversity and ecological functions is limited. Using novel and database-derived 16S rRNA gene and ribosomal protein sequences, we delineate the NS5 into 35 distinct species-level clusters, contained within four novel candidate genera. One candidate species, “Arcticimaribacter forsetii AHE01FL”, includes a novel cultured isolate, for which we provide a complete genome sequence—the first of an NS5—along with morphological insights using transmission electron microscopy. Assessing species’ spatial distribution dynamics across the Tara Oceans dataset, we identify depth as a key influencing factor, with 32 species preferring surface waters, as well as distinct patterns in relation to temperature, oxygen and salinity. Each species harbours a unique substrate-degradation potential along with predicted substrates conserved at the genus-level, e.g. alginate in NS5_F. Successional dynamics were observed for three species in a time-series dataset, likely driven by specialised substrate adaptations. We propose that the ecological niche partitioning of NS5 species is mainly based on specific abiotic factors, which define the niche space, and substrate availability that drive the species-specific temporal dynamics.

The impacts of climate change on the Arctic Ocean are manifesting throughout the ecosystem at an unprecedented rate. Of global importance are the impacts on heat and freshwater exchange between the Arctic and North Atlantic Oceans. An expanding Atlantic influence in the Arctic has accelerated sea-ice decline, weakened water column stability and supported the northward shift of temperate species. The only deep-water gateway connecting the Arctic and North Atlantic and thus, fundamental for these exchange processes is the Fram Strait. Previous research in this region is extensive, however, data on the ecology of microbial communities is limited, reflecting the wider bias towards temperate and tropical latitudes. Therefore, we present 14 metagenomes, 11 short-read from Illumina and three long-read from PacBio Sequel II, of the 0.2–3 µm fraction to help alleviate such biases and support future analyses on changing ecological patterns. Additionally, we provide 136 species-representative, manually refined metagenome-assembled genomes which can be used for comparative genomics analyses and addressing questions regarding functionality or distribution of taxa.

It is generally recognized that phages have a modulating role in the marine environment. Therefore, we hypothesized that phages can be a mortality factor for the dense heterotrophic bacterial population succeeding in phytoplankton blooms. For the marine carbon cycle, spring phytoplankton blooms are important recurring events. In this study, we focused onFlavobacteriia, because they are main responders during these blooms and have an important role in the degradation of polysaccharides. A cultivation-based approach was used, obtaining 44 lytic flavobacterial phages (flavophages), representing twelve new species from two viral realms –DuplodnaviriaandMonodnaviria. Taxonomic analysis allowed us to delineate ten new phage genera and seven new families, from which nine and four, respectively, had no previously cultivated representatives. Genomic analysis predicted various life styles and genomic replication strategies. A likely eukaryote-associated host habitat was reflected in the gene content of some of the flavophages. Detection in cellular metagenomes and by direct-plating indicated that part of these phages were actively replicating in the environment during the 2018 spring bloom. Furthermore, CRISPR/Cas spacers and re-isolation during two consecutive years indicated that, at least part of the new flavophages are stable components of the microbial community in the North Sea. Together, our results indicate that these diverse flavophages have the potential to modulate their respective host populations.

Marine algae annually sequester petagrams of carbon dioxide into polysaccharides, which are a central metabolic fuel for marine carbon cycling. Diatom microalgae produce sulfated polysaccharides containing methyl pentoses that are challenging to degrade for bacteria compared to other monomers, implicating these sugars as a potential carbon sink. Free-living bacteria occurring in phytoplankton blooms that specialise on consuming microalgal sugars, containing fucose and rhamnose remain unknown. Here, genomic and proteomic data indicate that small, coccoid, free-living Verrucomicrobiota specialise in fucose and rhamnose consumption during spring algal blooms in the North Sea. Verrucomicrobiota cell abundance was coupled with the algae bloom onset and accounted for up to 8% of the bacterioplankton. Glycoside hydrolases, sulfatases, and bacterial microcompartments, critical proteins for the consumption of fucosylated and sulfated polysaccharides, were actively expressed during consecutive spring bloom events. These specialised pathways were assigned to novel and discrete candidate species of the Akkermansiaceae and Puniceicoccaceae families, which we here describe as Candidatus Mariakkermansia forsetii and Candidatus Fucivorax forsetii. Moreover, our results suggest specialised metabolic pathways could determine the fate of complex polysaccharides consumed during algae blooms. Thus the sequestration of phytoplankton organic matter via methyl pentose sugars likely depend on the activity of specialised Verrucomicrobiota populations.

The flavobacterial genus Zobellia is considered as a model to study macroalgal polysaccharide degradation. The lack of data regarding its prevalence and abundance in coastal habitats constitutes a bottleneck to assess its ecological strategies. To overcome this issue, real-time quantitative PCR (qPCR) and fluorescence in situ hybridization (FISH) methods targeting the 16S rRNA gene were optimized to specifically detect and quantify Zobellia on the surface of diverse macroalgae. The newly designed qPCR primers and FISH probes targeted 98 and 100% of the Zobellia strains in silico and their specificity was confirmed using pure bacterial cultures. The dynamic range of the qPCR assay spanned 8 orders of magnitude from 10 to 108 16S rRNA gene copies and the detection limit was 0.01% relative abundance of Zobellia in environmental samples. Zobellia-16S rRNA gene copies were detected on all surveyed brown, green and red macroalgae, in proportion varying between 0.1 and 0.9% of the total bacterial copies. The absolute and relative abundance of Zobellia varied with tissue aging on the kelp Laminaria digitata. Zobellia cells were successfully visualized in Ulva lactuca and stranded Palmaria palmata surface biofilm using CARD-FISH, representing in the latter 10(5) Zobellia cells.cm(2) and 0.43% of total bacterial cells. Overall, qPCR and CARD-FISH assays enabled robust detection, quantification and localization of Zobellia representatives in complex samples, underlining their ecological relevance as primary biomass degraders potentially cross-feeding other microorganisms. (C) 2021 Elsevier GmbH. All rights reserved.

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.

The dynamics of diatoms and dinoflagellates have been monitored for many decades at the Helgoland Roads Long-Term Ecological Research site and are relatively well understood. In contrast, small-sized eukaryotic microbes and their community changes are still much more elusive, mainly due to their small size and uniform morphology, which makes them difficult to identify microscopically. By using next-generation sequencing, we wanted to shed light on the Helgoland planktonic community dynamics, including nano- and picoplankton, during a spring bloom. We took samples from March to May 2016 and sequenced the V4 region of the 18S rDNA. Our results showed that mixotrophic and heterotrophic taxa were more abundant than autotrophic diatoms. Dinoflagellates dominated the sequence assemblage, and several small-sized eukaryotic microbes like Haptophyta, Choanoflagellata, Marine Stramenopiles and Syndiniales were identified. A diverse background community including taxa from all size classes was present during the whole sampling period. Five phases with several communities were distinguished. The fastest changes in community composition took place in phase 3, while the communities from phases 1 to 5 were more similar to each other despite contrasting environmental conditions. Synergy effects of next-generation sequencing and traditional methods may be exploited in future long-term observations.

Microbial degradation of algal biomass following spring phytoplankton blooms has been characterised as a concerted effort among multiple clades of heterotrophic bacteria. Despite their significance to overall carbon turnover, many of these clades have resisted cultivation. One clade known from 16S rRNA gene sequencing surveys at Helgoland in the North Sea, was formerly identified as belonging to the genus Ulvibacter. This clade rapidly responds to algal blooms, transiently making up as much as 20% of the free-living bacterioplankton. Sequence similarity below 95% between the 16S rRNA genes of described Ulvibacter species and those from Helgoland suggest this is a novel genus. Analysis of 40 metagenome assembled genomes (MAGs) derived from samples collected during spring blooms at Helgoland support this conclusion. These MAGs represent three species, only one of which appears to bloom in response to phytoplankton. MAGs with estimated completeness greater than 90% could only be recovered for this abundant species. Additional, less complete, MAGs belonging to all three species were recovered from a mini-metagenome of cells sorted via flow cytometry using the genus specific ULV995 fluorescent rRNA probe. Metabolic reconstruction indicates this highly abundant species most likely degrades proteins and the polysaccharide laminarin. Fluorescence in situ hybridisation showed coccoid cells, with a mean diameter of 0.78mm, with standard deviation of 0.12μm. Based on the phylogenetic and genomic characteristics of this clade, we propose the novel candidate genus Candidatus Prosiliicoccus, and for the most abundant and well characterised of the three species the name Candidatus Prosiliicoccus vernus.

On-going studies of phytoplankton-bacterioplankton interactions at the long-term ecological research site Helgoland Roads have indicated that many of the heterotrophic bacterial taxa have not yet been cultivated. A high-throughput approach combining whole cell matrix-assisted laser desorption ionization – time of flight mass spectroscopy with 16S rRNA gene sequencing was applied to the spring bloom of 2016. Aiming at an assessment of cultivability during a spring bloom, cultivation on solid marine media had to be used since dilution to extinction would not have been feasible for a high-throughput approach, as performed in this study. A total of 5023 isolates were obtained from nine weekly samples on eight different solid media between the early-bloom and post-bloom periods. Most of the 4136 strains identified affiliated with Bacteroidetes (13.3%), Gammaproteobacteria (26.9%), Alphaproteobacteria (40.6%) and Actinobacteria (6.7%). Of the 271 operational phylogenetic units (OPUs) identified, 13 are likely to represent novel genera and 143 novel species. A comparison with 16S rRNA gene tag data indicated that most of the isolates were rather rare in surface waters, with the exception of five OPUs affiliating with Rhodobacteraceae, Polaribacter, Psychromonas and Pseudoalteromonas. The effort yielded many novel isolates, yet most of the abundant heterotrophic bacteria still remained elusive. The large strain collection obtained will not only provide insights into the succession of the cultivable fraction of the bacterioplankton, but also enable fine-tuned taxonomic and physiological follow-up studies for improving our knowledge on heterotrophic bacteria in North Sea waters., This study was funded by the Max Planck Society and supported by the Deutsche Forschungsgemeinschaft (DFG) within the framework of research unit FOR2406 ‘Proteogenomics of Marine Polysaccharide Utilization (POMPU)’ by a grant to RA (AM 73/9-1), and by the Spanish Ministry of Economy projects CLG2015_66686-C3-1-P and PGC2018-096956-B-C41, both also supported by the European Regional Development Fund (FEDER). The authors thank the University of the Balearic Islands for the MALDI-TOF MS spectra, and Thomas Ben Francis for bioinformatic support.

Este artículo contiene 6 páginas, 4 figuras, 1 tabla, The Thaumarchaeota SAGMCG-1 group and, in particular, members of the genus Nitrosotalea have highoccurrence in acidic soils, the rhizosphere, groundwater and oligotrophic lakes, and play a potential rolein nitrogen cycling. In this study, the specific oligonucleotide fluorescence in situ hybridization probeSAG357 was designed for this Thaumarchaeota group based on the available 16S rRNA gene sequencesin databases, and included the ammonia-oxidizing species Nitrosotalea devanaterra. Cell permeabiliza-tion for catalyzed reporter deposition fluorescence in situ detection and the hybridization conditionswere optimized on enrichment cultures of the target species N. devanaterra, as well as the non-targetammonia-oxidizing archaeon Nitrosopumilus maritimus. Probe specificity was improved with a competi-tor oligonucleotide, and fluorescence intensity and cell visualization were enhanced by the design andapplication of two adjacent helpers. Probe performance was tested in soil samples along a pH gradient,and counting results matched the expected in situ distributions. Probe SAG357 and the CARD-FISH pro-tocol developed in the present study will help to improve the current understanding of the ecology andphysiology of N. devanaterra and its relatives in natural environments., This research was supported by grantsPIRENA-CGL2009-13318 and DARKNESS-CGL2012-32747 from theSpanish Office of Science (MINECO) to EOC, and by a Spanish FPIpre-doctoral scholarship (MINECO-PIRENA) to CRO. JBC and BFacknowledge funding from the Max Planck Society.

The N2 -fixing (diazotrophic) community in marine ecosystems is dominated by non-cyanobacterial microorganisms. Yet, very little is known about their identity, function and ecological relevance due to a lack of cultured representatives. Here we report a novel heterotrophic diazotroph isolated from the oxygen minimum zone (OMZ) off Peru. The new species belongs to the genus Sagittula (Rhodobacteraceae, Alphaproteobacteria) and its capability to fix N2 was confirmed in laboratory experiments. Genome sequencing revealed that it is a strict heterotroph with a high versatility in substrate utilization and energy acquisition mechanisms. Pathways for sulfide oxidation and nitrite reduction to nitrous oxide are encoded in the genome and might explain the presence throughout the Peruvian OMZ. The genome further indicates that this novel organism could be in direct interaction with other microbes or particles. NanoSIMS analyses were used to compare the metabolic potential of S. castanea with single-cell activity in situ; however, N2 fixation by this diazotroph could not be detected at the isolation site. While the biogeochemical impact of S. castanea is yet to be resolved, its abundance and widespread distribution suggests that its potential to contribute to the marine N input could be significant at a larger geographical scale.

Gammaproteobacterial Reinekea spp. were detected during North Sea spring algae blooms in the years 2009-2012, with relative abundances of up to 16% in the bacterioplankton. Here, we explore the ecophysiology of 'R. forsetii' strain Hel1_31_D35 that was isolated during the 2010 spring bloom using (i) its manually annotated, high-quality closed genome, (ii) re-analysis of in situ data from the 2009-2012 blooms and (iii) physiological tests. High resolution analysis of 16S rRNA gene sequences suggested that 'R. forsetii' dominated Reinekea populations during these blooms. This was corroborated by retrieval of almost complete Hel1_31_D35 genomes from 2009 and 2010 bacterioplankton metagenomes. Strain Hel1_31_D35 can use numerous low-molecular weight substrates including diverse sugar monomers, and few but relevant algal polysaccharides such as mannan, α-glucans, and likely bacterial peptidoglycan. It oxidizes thiosulfate to sulfate, and ferments under anoxic conditions. The strain can attach to algae and thrives at low phosphate concentrations as they occur during blooms. Its genome encodes RTX toxin and secretion proteins, and in cultivation experiments Hel1_31_D35 crude cell extracts inhibited growth of a North Sea Polaribacter strain. Our data suggest that the combination of these traits make strain Hel1_31_D35 a versatile opportunist that is particularly competitive during spring phytoplankton blooms.

Coastal phytoplankton blooms are frequently followed by successive blooms of heterotrophic bacterial clades. The class Flavobacteriia within the Bacteroidetes has been shown to play an important role in the degradation of high molecular weight substrates that become available in the later stages of such blooms. One of the flavobacterial clades repeatedly observed over the course of several years during phytoplankton blooms off the coast of Helgoland, North Sea, is Vis6. This genus-level Glade belongs to the family Cryomorphaceae and has been resistant to cultivation to date. Based on metagenome assembled genomes, comparative 16S rRNA gene sequence analyses and fluorescence in situ hybridization, we here propose a novel candidate genus Abditibacter, comprising three novel species Candidatus Abditibacter vernus, Candidatus Abditibacter forsetii and Candidatus Abditibacter autumni. While the small genomes of the three novel photoheterotrophic species encode highly similar gene repertoires, including genes for degradation of proteins and algal storage polysaccharides such as laminarin, two of them - Ca. A. vernus and Ca. A. forsetii - seem to have a preference for spring blooms, while Ca. A. autumni almost exclusively occurs in late summer and autumn. (C) 2020 The Author(s). Published by Elsevier GmbH.

SummarySpring phytoplankton blooms in temperate environments contribute disproportionately to global marine productivity. Bloom-derived organic matter, much of it occurring as polysaccharides, fuels biogeochemical cycles driven by interacting autotrophic and heterotrophic communities. We tracked changes in the mode of polysaccharide utilization by heterotrophic bacteria during the course of a diatom-dominated bloom in the German Bight, North Sea. Polysaccharides can be taken up in a ‘selfish’ mode, where initial hydrolysis is coupled to transport into the periplasm, such that little to no low molecular weight (LMW) products are externally released to the environment. Alternatively, polysaccharides hydrolyzed by cell-surface attached or free extracellular enzymes (external hydrolysis) yield LMW products available to the wider bacterioplankton community. In the early bloom phase, selfish activity was accompanied by low extracellular hydrolysis rates of a few polysaccharides. As the bloom progressed, selfish uptake increased markedly, and external hydrolysis rates increased, but only for a limited range of substrates. The late bloom phase was characterized by high external hydrolysis rates of a broad range of polysaccharides, and reduced selfish uptake of polysaccharides, except for laminarin. Substrate utilization mode is related both to substrate structural complexity and to the bloom-stage dependent composition of the heterotrophic bacterial community.Originality statementThe means by which heterotrophic bacteria cooperate and compete to obtain substrates is a key factor determining the rate and location at which organic matter is cycled in the ocean. Much of this organic matter is high molecular weight (HMW), and must be enzymatically hydrolyzed to smaller pieces to be processed by bacterial communities. Some of these enzyme-producing bacteria are ‘selfish’, processing HMW organic matter without releasing low molecular weight (LMW) products to the environment. Other bacteria hydrolyze HMW substrates in a manner that releases LMW products to the wider bacterial community. How these mechanisms of substrate hydrolysis work against a changing background of organic matter supply is unclear. Here, we measured changing rates and mechanisms of substrate processing during the course of a natural phytoplankton bloom in the North Sea. Selfish bacteria generally dominate in the initial bloom stages, but a greater supply of increasingly complex substrates in later bloom stages leads to external hydrolysis of a wider range of substrates, increasing the supply of LMW hydrolysis products to the wider bacterial community.

Primary productivity occurs throughout the deep euphotic zone of the oligotrophic South Pacific Gyre (SPG), fueled largely by the regeneration of nutrients and thus recycling of organic matter. We investigated the heterotrophic capabilities of the SPG’s bacterial communities by examining their ability to process polysaccharides, an important component of marine organic matter. We focused on the initial step of organic matter degradation by measuring the activities of extracellular enzymes that hydrolyze six different polysaccharides to smaller sizes. This process can occur by two distinct mechanisms: “selfish uptake,” in which initial hydrolysis is coupled to transport of large polysaccharide fragments into the periplasmic space of bacteria, with little to no loss of hydrolysis products to the external environment, and “external hydrolysis,” in which low molecular weight (LMW) hydrolysis products are produced in the external environment. Given the oligotrophic nature of the SPG, we did not expect high enzymatic activity; however, we found that all six polysaccharides were hydrolyzed externally and taken up selfishly in the central SPG, observations that may be linked to a comparatively high abundance of diatoms at the depth and location sampled (75 m). At the edge of the gyre and close to the center of the gyre, four of six polysaccharides were externally hydrolyzed, and a lower fraction of the bacterial community showed selfish uptake. One polysaccharide (fucoidan) was selfishly taken up without measurable external hydrolysis at two stations. Additional incubations of central gyre water from depths of 1,250 and 2,800 m with laminarin (an abundant polysaccharide in the ocean) led to extreme growth of opportunistic bacteria (Alteromonas), as tracked by cell counts and next generation sequencing of the bacterial communities. These Alteromonas appear to concurrently selfishly take up laminarin and release LMW hydrolysis products. Overall, extracellular enzyme activities in the SPG were similar to activities in non-oligotrophic regions, and a considerable fraction of the community was capable of selfish uptake at all three stations. A diverse set of bacteria responded to and are potentially important for the recycling of organic matter in the SPG.

Seamounts, often rising hundreds of metres above surrounding seafloor, obstruct the flow of deep-ocean water. While the retention of deep-water by seamounts is predicted from ocean circulation models, its empirical validation has been hampered by large scale and slow rate of the interaction. To overcome these limitations we use the growth of planktonic bacteria to assess the retention time of deep-ocean water by a seamount. The selected Tropic Seamount in the North-Eastern Atlantic is representative for the majority of isolated seamounts, which do not affect the surface ocean waters. We prove deep-water is retained by the seamount by measuring 2.4× higher bacterial concentrations in the seamount-associated or ‘sheath’-water than in deep-ocean water unaffected by seamounts. Genomic analyses of flow-sorted, dominant sheath-water bacteria confirm their planktonic origin, whilst proteomic analyses of the sheath-water bacteria, isotopically labelled in situ, indicate their slow growth. According to our radiotracer experiments, it takes the sheath-water bacterioplankton 1.5 years to double their concentration. Therefore, the seamount should retain the deep-ocean water for 1.8 years for the deep-ocean bacterioplankton to grow to the 2.4× higher concentration in the sheath-water. We propose that turbulent mixing of the seamount sheath-water stimulates bacterioplankton growth by increasing cell encounter rate with ambient dissolved organic molecules.

Jellyfi sh blooms have increased worldwide, and the outbreaks of jellyfi sh population not only aff ect the food web structures via voracious predation but also play an important role in the dynamics of nutrients and oxygen in planktonic food webs. However, it remains unclear whether specifi c carbon compounds released through jellyfi sh metabolic processes have the potential to shape bacterial community composition. Therefore, in this study, we aimed to investigate the compositional succession of the bacterioplankton community in response to the dissolved organic matter (DOM) released by the live Scyphomedusae Cyanea lamarckii and Chrysaora hysoscella collected from Helgoland Roads of the North Sea. The bacterial community was signifi cantly stimulated by the DOM released form live jellyfish and different dominant phylotypes were observed for these two Scyphomedusae species. Furthermore, the bacterial community structures in the diff erent DOM sources, jellyfi sh-incubated media, Kabeltonne seawater, and artificial seawater (DOM-free) were signifi cantly diff erent, as revealed by automated ribosomal intergenic spacer analysis fingerprints. Catalyzed reporter deposition fl uorescence in situ hybridization (CARD-FISH) revealed a rapid species-specific shift in bacterial community composition. Gammaproteobacteria dominated the community instead of the Bacteroidetes community for C . lamarckii, whereas Gammaproteobacteria and Bacteroidetes dominated the community for C . hysoscella. The signifi cant diff erences in the bacterial community composition and succession indicate that the components of the DOM released by jellyfish might differ with jellyfish species.

One of the most abundant sources of organic carbon in the ocean is glycolate, the secretion of which by marine phytoplankton results in an estimated annual flux of one petagram of glycolate in marine environments

Although fluorescence in situ hybridization (FISH) with specific ribosomal RNA (rRNA) targeted oligonucleotides is a standard method to detect and identify microorganisms, the specific detection of genes in bacteria and archaea, for example by using geneFISH, requires complicated and lengthy (> 30 h) procedures. Here we report a much improved protocol, direct-geneFISH, which allows specific gene and rRNA detection within less than 6 h. For direct-geneFISH, CARD steps are removed and fluorochrome-labeled polynucleotide gene probes and rRNA-targeted oligonucleotide probes are hybridized simultaneously. The protocol allows quantification of gene copy numbers per cell and the signal of the directly labeled probes enables a sub-cellular localization of the rRNA and target gene. The detection efficiencies of direct-geneFISH were first evaluated on Escherichia coli carrying the target gene on a copy-control vector. We could show that gene copy numbers correlated to the geneFISH signal within the cells. The new protocol was then applied for the detection of the sulfate thiolhydrolase (soxB) genes in cells of the gammaproteobacterial clade SUP05 in Lake Rogoznica, Croatia. Cell and gene detection efficiencies by direct-geneFISH were statistically identical to those obtained with the original geneFISH, demonstrating the suitability of the simpler and faster protocol for environmental samples. This article is protected by copyright. All rights reserved.

Marine Bacteroidetes have pronounced capabilities of degrading high molecular weight organic matter such as proteins and polysaccharides. Previously we reported on 76 Bacteroidetes-affiliated fosmids from the North Atlantic Ocean's boreal polar and oligotrophic subtropical provinces. Here, we report on the analysis of further 174 fosmids from the same libraries. The combined, re-assembled dataset (226 contigs; 8.8 Mbp) suggests that planktonic Bacteroidetes at the oligotrophic southern station use more peptides and bacterial and animal polysaccharides, whereas Bacteroidetes at the polar station (East-Greenland Current) use more algal and plant polysaccharides. The latter agrees with higher abundances of algae and terrigenous organic matter, including plant material, at the polar station. Results were corroborated by in-depth bioinformatic analysis of 14 polysaccharide utilisation loci from both stations, suggesting laminarin-specificity for four and specificity for sulfated xylans for two loci. In addition, one locus from the polar station supported use of non-sulfated xylans and mannans, possibly of plant origin. While peptides likely represent a prime source of carbon for Bacteroidetes in open oceans, our data suggest that as yet unstudied clades of these Bacteroidetes have a surprisingly broad capacity for polysaccharide degradation. In particular, laminarin-specific PULs seem widespread and thus must be regarded as globally important.

Marine sediments are the largest carbon sink on earth. Nearly half of dark carbon fixation in the oceans occurs in coastal sediments, but the microorganisms responsible are largely unknown. By integrating the 16S rRNA approach, single-cell genomics, metagenomics and transcriptomics with (14)C-carbon assimilation experiments, we show that uncultured Gammaproteobacteria account for 70-86% of dark carbon fixation in coastal sediments. First, we surveyed the bacterial 16S rRNA gene diversity of 13 tidal and sublittoral sediments across Europe and Australia to identify ubiquitous core groups of Gammaproteobacteria mainly affiliating with sulfur-oxidizing bacteria. These also accounted for a substantial fraction of the microbial community in anoxic, 490-cm-deep subsurface sediments. We then quantified dark carbon fixation by scintillography of specific microbial populations extracted and flow-sorted from sediments that were short-term incubated with (14)C-bicarbonate. We identified three distinct gammaproteobacterial clades covering diversity ranges on family to order level (the Acidiferrobacter, JTB255 and SSr clades) that made up >50% of dark carbon fixation in a tidal sediment. Consistent with these activity measurements, environmental transcripts of sulfur oxidation and carbon fixation genes mainly affiliated with those of sulfur-oxidizing Gammaproteobacteria. The co-localization of key genes of sulfur and hydrogen oxidation pathways and their expression in genomes of uncultured Gammaproteobacteria illustrates an unknown metabolic plasticity for sulfur oxidizers in marine sediments. Given their global distribution and high abundance, we propose that a stable assemblage of metabolically flexible Gammaproteobacteria drives important parts of marine carbon and sulfur cycles.

The dynamics of diatoms and dinoflagellates have been monitored for many decades at the Helgoland Roads Long-Term Ecological Research site and are relatively well understood. In contrast, small-sized eukaryotic microbes and their community changes are still much more elusive, mainly due to their small size and uniform morphology, which makes them difficult to identify microscopically. By using next-generation sequencing, we wanted to shed light on the Helgoland planktonic community dynamics, including nano- and picoplankton, during a spring bloom. We took samples from March to May 2016 and sequenced the V4 region of the 18S rDNA. Our results showed that mixotrophic and heterotrophic taxa were more abundant than autotrophic diatoms. Dinoflagellates dominated the sequence assemblage, and several small-sized eukaryotic microbes like Haptophyta, Choanoflagellata, Marine Stramenopiles and Syndiniales were identified. A diverse background community including taxa from all size classes was present during the whole sampling period. Five phases with several communities were distinguished. The fastest changes in community composition took place in phase 3, while the communities from phases 1 to 5 were more similar to each other despite contrasting environmental conditions. Synergy effects of next-generation sequencing and traditional methods may be exploited in future long-term observations.

The South Pacific Gyre (SPG) covers 10% of the ocean's surface and is often regarded as a marine biological desert. To gain an on-site overview of the remote, ultra-oligotrophic microbial community of the SPG we developed a novel on-board analysis pipeline, which combines next-generation sequencing with fluorescence in situ hybridisation and automated cell enumeration. We tested the pipeline during the SO245 "UltraPac" cruise from Chile to New Zealand and found that the overall microbial community of the SPG was highly similar to that of other oceanic gyres. The SPG was dominated by 20 major bacterial clades, including SAR11, SAR116, AEGEAN-169 marine group, SAR86, Prochlorococcus, SAR324, SAR406, and SAR202. Most of the bacterial clades showed a strong vertical (20 m - 5000 m), but only a weak longitudinal (80°W - 160°W), distribution pattern. Surprisingly, in the central gyre Prochlorococcus, the dominant photosynthetic organism, had only low cellular abundances in the upper waters (20 - 80 m) and were more frequent around the 1% irradiance zone (100 - 150 m). Instead, the surface waters of the central gyre were dominated by SAR11, SAR86, and SAR116 clades known to harbour light-driven proton pumps. The alphaproteobacterial AEGEAN-169 marine-group was particularly abundant in the surface waters of the central gyre indicating a potentially interesting adaptation to ultraoligotrophic waters and high solar irradiance. In the future, the newly developed community analysis pipeline will allow for on-site insights into a microbial community within 35 hours of sampling, which will permit more targeted sampling efforts and hypothesis-driven research.ImportanceThe South Pacific Gyre is due to its vast size and remoteness one of the least studied oceanic regions on earth. However, both remote sensing and in situ measurements indicated that the activity of its microbial community contributes significantly to global biogeochemical cycles. Presented here is an unparalleled investigation of the microbial community of the SPG from 20 - 5000 m depth covering a geographic distance of 7000 km. This insight was achieved through the development of a novel on-board analysis pipeline, which combines next-generation sequencing with fluorescence in situ hybridisation and automated cell enumeration. The pipeline is well comparable to on-shore systems based on the Illumina platforms and yields microbial community data in less than 35 hours after sampling. Going forward the ability to gain on-site knowledge of a remote microbial community will permit hypothesis-driven research, through the generation of novel scientific questions and subsequent additional targeted sampling efforts. Copyright © 2019 American Society for Microbiology.

Identifying the roles played by individual heterotrophic bacteria in the degradation of high molecular weight (HMW) substrates is critical to understanding the constraints on carbon cycling in the ocean. At five sites in the Atlantic Ocean, we investigated the processing of organic matter by tracking changes in microbial community composition as HMW polysaccharides were enzymatically hydrolysed over time. During this investigation, we discovered that a considerable fraction of heterotrophic bacteria uses a newly-identified ‘selfish’ mode of substrate processing. We therefore additionally examined the balance of individual substrate utilisation mechanisms at different locations by linking individual microorganisms to distinct substrate utilisation mechanisms. Through FISH and uptake of fluorescently-labelled polysaccharides, ‘selfish’ organisms were identified as belonging to the Bacteroidetes, Planctomycetes and Gammaproteobacteria. ‘Sharing’ (extracellular enzyme producing) and ‘scavenging’ (non-enzyme producing) organisms predominantly belonged to the Alteromonadaceae and SAR11 clades, respectively. The extent to which individual mechanisms prevail depended on the initial population structure of the bacterial community at a given location and time, as well as the growth rate of specific bacteria. Furthermore, the same substrate was processed in different ways by different members of a pelagic microbial community, pointing to significant follow-on effects for carbon cycling.

Fluorescence in situ hybridization (FISH) has become a vital tool for environmental and medical microbiology and is commonly used for the identification, localization, and isolation of defined microbial taxa. However, fluorescence signal strength is often a limiting factor for targeting all members in a microbial community. Here, we present the application of a multilabeled FISH approach (MiL-FISH) that (i) enables the simultaneous targeting of up to seven microbial groups using combinatorial labeling of a single oligonucleotide probe, (ii) is applicable for the isolation of unfixed environmental microorganisms via fluorescence-activated cell sorting (FACS), and (iii) improves signal and imaging quality of tissue sections in acrylic resin for precise localization of individual microbial cells. We show the ability of MiL-FISH to distinguish between seven microbial groups using a mock community of marine organisms and its applicability for the localization of bacteria associated with animal tissue and their isolation from host tissues using FACS. To further increase the number of potential target organisms, a streamlined combinatorial labeling and spectral imaging-FISH (CLASI-FISH) concept with MiL-FISH probes is presented here. Through the combination of increased probe signal, the possibility of targeting hard-to-detect taxa and isolating these from an environmental sample, the identification and precise localization of microbiota in host tissues, and the simultaneous multilabeling of up to seven microbial groups, we show here that MiL-FISH is a multifaceted alternative to standard monolabeled FISH that can be used for a wide range of biological and medical applications.