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To understand how extreme halophiles respond to recurrent disturbances, we challenged the communities thriving in salt-saturated (~36% salts) ~230 L brine mesocosms to repeated dilutions down to 13% (D13 mesocosm) or 20% (D20 mesocosm) salts each time mesocosms reached salt saturation due to evaporation (for 10 and 17 cycles, respectively) over 813 days. Depending on the magnitude of dilution, the most prevalent species, Haloquadratum walsbyi and Salinibacter ruber, either increased in dominance by replacing less competitive populations (for D20, moderate stress conditions), or severely decreased in abundance and were eventually replaced by other congeneric species better adapted to the higher osmotic stress (for D13, strong stress conditions). Congeneric species replacement was commonly observed within additional abundant genera in response to changes in environmental or biological conditions (e.g. phage predation) within the same system and under a controlled perturbation of a relevant environmental parameter. Therefore, a genus is an ecologically important level of diversity organization, not just a taxonomic rank, that persists in the environment based on congeneric species replacement due to relatively high functional overlap (gene sharing), with important consequences for the success of the lineage, and similar to the success of a species via strain-replacement. Further, our results showed that successful species were typically accompanied by the emergence of their own viral cohorts, whose intra-cohort diversity appeared to strongly covary with, and likely drive, the intra-host diversity. Collectively, our results show that brine communities are ecologically resilient and continuously adapting to changing environments by transitioning to alternative stable states., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

One of the most hostile marine habitats on Earth is the surface of the South Pacific Gyre (SPG), characterized by high solar radiation, extreme nutrient depletion, and low productivity. During the SO-245 "UltraPac" cruise through the center of the ultra-oligotrophic SPG, the marine alphaproteobacterial group AEGEAN169 was detected by fluorescence in situ hybridization at relative abundances up to 6% of the total microbial community in the uppermost water layer, with two distinct populations (Candidatus Nemonibacter and Ca. Indicimonas). The high frequency of dividing cells combined with high transcript levels suggests that both clades may be highly metabolically active. Comparative metagenomic and metatranscriptomic analyses of AEGEAN169 revealed that they encoded subtle but distinct metabolic adaptions to this extreme environment in comparison to their competitors SAR11, SAR86, SAR116, and Prochlorococcus. Both AEGEAN169 clades had the highest percentage of transporters per predicted proteins (9.5% and 10.6%, respectively). In particular, the high expression of ABC transporters in combination with proteorhodopsins and the catabolic pathways detected suggest a potential scavenging lifestyle for both AEGEAN169 clades. Although both AEGEAN169 clades may share the genomic potential to utilize phosphonates as a phosphorus source, they differ in their metabolic pathways for carbon and nitrogen. Ca. Nemonibacter potentially use glycine-betaine, whereas Ca. Indicimonas may catabolize urea, creatine, and fucose. In conclusion, the different potential metabolic strategies of both clades suggest that both are well adapted to thrive resource-limited conditions and compete well with other dominant microbial clades in the uppermost layers of SPG surface waters., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

The Arctic Ocean is experiencing unprecedented changes because of climate warming, necessitating detailed analyses on the ecology and dynamics of biological communities to understand current and future ecosystem shifts. Here, we generated a four-year, high-resolution amplicon dataset along with one annual cycle of PacBio HiFi read metagenomes from the East Greenland Current (EGC), and combined this with datasets spanning different spatiotemporal scales (Tara Arctic and MOSAiC) to assess the impact of Atlantic water influx and sea-ice cover on bacterial communities in the Arctic Ocean. Densely ice-covered polar waters harboured a temporally stable, resident microbiome. Atlantic water influx and reduced sea-ice cover resulted in the dominance of seasonally fluctuating populations, resembling a process of "replacement" through advection, mixing and environmental sorting. We identified bacterial signature populations of distinct environmental regimes, including polar night and high-ice cover, and assessed their ecological roles. Dynamics of signature populations were consistent across the wider Arctic; e.g. those associated with dense ice cover and winter in the EGC were abundant in the central Arctic Ocean in winter. Population- and community-level analyses revealed metabolic distinctions between bacteria affiliated with Arctic and Atlantic conditions; the former with increased potential to use bacterial- and terrestrial-derived substrates or inorganic compounds. Our evidence on bacterial dynamics over spatiotemporal scales provides novel insights into Arctic ecology and indicates a progressing Biological Atlantification of the warming Arctic Ocean, with consequences for food webs and biogeochemical cycles., (© 2023. The Author(s).)

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., (© 2022. The Author(s).)

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., (© 2021. The Author(s).)

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., (© 2021. The Author(s).)

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., (© 2021. The Author(s).)

Massive releases of organic substrates during marine algal blooms trigger growth of many clades of heterotrophic bacteria. Algal polysaccharides represent the most diverse and structurally complex class of these substrates, yet their role in shaping the microbial community composition is poorly understood. We investigated, whether polysaccharide utilization capabilities contribute to niche differentiation of Polaribacter spp. (class Flavobacteriia; known to include relevant polysaccharide-degraders) that were abundant during 2009-2012 spring algal blooms in the southern North Sea. We identified six distinct Polaribacter clades using phylogenetic and phylogenomic analyses, quantified their abundances via fluorescence in situ hybridization, compared metagenome-assembled genomes, and assessed in situ gene expression using metaproteomics. Four clades with distinct polysaccharide niches were dominating. Polaribacter 2-a comprised typical first responders featuring small genomes with limited polysaccharide utilization capacities. Polaribacter 3-a were abundant only in 2010 and possessed a distinct sulfated α-glucoronomannan degradation potential. Polaribacter 3-b responded late in blooms and had the capacity to utilize sulfated xylan. Polaribacter 1-a featured high numbers of glycan degradation genes and were particularly abundant following Chattonella algae blooms. These results support the hypothesis that sympatric Polaribacter clades occupy distinct glycan niches during North Sea spring algal blooms.

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.

Heterotrophic microbial communities process much of the carbon fixed by phytoplankton in the ocean, thus having a critical role in the global carbon cycle. A major fraction of the phytoplankton-derived substrates are high-molecular-weight (HMW) polysaccharides. For bacterial uptake, these substrates must initially be hydrolysed to smaller sizes by extracellular enzymes. We investigated polysaccharide hydrolysis by microbial communities during a transect of the Atlantic Ocean, and serendipitously discovered-using super-resolution structured illumination microscopy-that up to 26% of total cells showed uptake of fluorescently labelled polysaccharides (FLA-PS). Fluorescence in situ hybridisation identified these organisms as members of the bacterial phyla Bacteroidetes and Planctomycetes and the gammaproteobacterial genus Catenovulum. Simultaneous membrane staining with nile red indicated that the FLA-PS labelling occurred in the cell but not in the cytoplasm. The dynamics of FLA-PS staining was further investigated in pure culture experiments using Gramella forsetii, a marine member of Bacteroidetes. The staining patterns observed in environmental samples and pure culture tests are consistent with a 'selfish' uptake mechanisms of larger oligosaccharides (>600 Da), as demonstrated for gut Bacteroidetes. Ecologically, this alternative polysaccharide uptake mechanism secures substantial quantities of substrate in the periplasmic space, where further processing can occur without diffusive loss. Such a mechanism challenges the paradigm that hydrolysis of HMW substrates inevitably yields low-molecular-weight fragments that are available to the surrounding community and demonstrates the importance of an alternative mechanism of polysaccharide uptake in marine bacteria.

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.

Symbiotic relationships between phytoplankton and N2-fixing microorganisms play a crucial role in marine ecosystems. The abundant and widespread unicellular cyanobacteria group A (UCYN-A) has recently been found to live symbiotically with a haptophyte. Here, we investigated the effect of nitrogen (N), phosphorus (P), iron (Fe) and Saharan dust additions on nitrogen (N2) fixation and primary production by the UCYN-A-haptophyte association in the subtropical eastern North Atlantic Ocean using nifH expression analysis and stable isotope incubations combined with single-cell measurements. N2 fixation by UCYN-A was stimulated by the addition of Fe and Saharan dust, although this was not reflected in the nifH expression. CO2 fixation by the haptophyte was stimulated by the addition of ammonium nitrate as well as Fe and Saharan dust. Intriguingly, the single-cell analysis using nanometer scale secondary ion mass spectrometry indicates that the increased CO2 fixation by the haptophyte in treatments without added fixed N is likely an indirect result of the positive effect of Fe and/or P on UCYN-A N2 fixation and the transfer of N2-derived N to the haptophyte. Our results reveal a direct linkage between the marine carbon and nitrogen cycles that is fuelled by the atmospheric deposition of dust. The comparison of single-cell rates suggests a tight coupling of nitrogen and carbon transfer that stays balanced even under changing nutrient regimes. However, it appears that the transfer of carbon from the haptophyte to UCYN-A requires a transfer of nitrogen from UCYN-A. This tight coupling indicates an obligate symbiosis of this globally important diazotrophic association.

Subtropical oceanic gyres are the most extensive biomes on Earth where SAR11 and Prochlorococcus bacterioplankton numerically dominate the surface waters depleted in inorganic macronutrients as well as in dissolved organic matter. In such nutrient poor conditions bacterioplankton could become photoheterotrophic, that is, potentially enhance uptake of scarce organic molecules using the available solar radiation to energise appropriate transport systems. Here, we assessed the photoheterotrophy of the key microbial taxa in the North Atlantic oligotrophic gyre and adjacent regions using (33)P-ATP, (3)H-ATP and (35)S-methionine tracers. Light-stimulated uptake of these substrates was assessed in two dominant bacterioplankton groups discriminated by flow cytometric sorting of tracer-labelled cells and identified using catalysed reporter deposition fluorescence in situ hybridisation. One group of cells, encompassing 48% of all bacterioplankton, were identified as members of the SAR11 clade, whereas the other group (24% of all bacterioplankton) was Prochlorococcus. When exposed to light, SAR11 cells took 31% more ATP and 32% more methionine, whereas the Prochlorococcus cells took 33% more ATP and 34% more methionine. Other bacterioplankton did not demonstrate light stimulation. Thus, the SAR11 and Prochlorococcus groups, with distinctly different light-harvesting mechanisms, used light equally to enhance, by approximately one-third, the uptake of different types of organic molecules. Our findings indicate the significance of light-driven uptake of essential organic nutrients by the dominant bacterioplankton groups in the surface waters of one of the less productive, vast regions of the world's oceans-the oligotrophic North Atlantic subtropical gyre.

Next-generation sequencing (NGS) technologies have enabled the application of broad-scale sequencing in microbial biodiversity and metagenome studies. Biodiversity is usually targeted by classifying 16S ribosomal RNA genes, while metagenomic approaches target metabolic genes. However, both approaches remain isolated, as long as the taxonomic and functional information cannot be interrelated. Techniques like self-organizing maps (SOMs) have been applied to cluster metagenomes into taxon-specific bins in order to link biodiversity with functions, but have not been applied to broad-scale NGS-based metagenomics yet. Here, we provide a novel implementation, demonstrate its potential and practicability, and provide a web-based service for public usage. Evaluation with published data sets mimicking varyingly complex habitats resulted into classification specificities and sensitivities of close to 100% to above 90% from phylum to genus level for assemblies exceeding 8 kb for low and medium complexity data. When applied to five real-world metagenomes of medium complexity from direct pyrosequencing of marine subsurface waters, classifications of assemblies above 2.5 kb were in good agreement with fluorescence in situ hybridizations, indicating that biodiversity was mostly retained within the metagenomes, and confirming high classification specificities. This was validated by two protein-based classifications (PBCs) methods. SOMs were able to retrieve the relevant taxa down to the genus level, while surpassing PBCs in resolution. In order to make the approach accessible to a broad audience, we implemented a feature-rich web-based SOM application named TaxSOM, which is freely available at http://www.megx.net/toolbox/taxsom. TaxSOM can classify reads or assemblies exceeding 2.5 kb with high accuracy and thus assists in linking biodiversity and functions in metagenome studies, which is a precondition to study microbial ecology in a holistic fashion.

Members of the class Flavobacteria in the phylum Bacteroidetes are among the most abundant picoplankton in coastal and polar oceans. Their diversity is high in marine waters. However, quantitative information about distribution patterns of flavobacterial clades is scarce. We analyzed the diversity and clade-specific abundances of individual Flavobacteria in different oceanic provinces in the North Atlantic Ocean. Samples were taken along the 30 degrees W meridian between the East Greenland current and the North Atlantic subtropical gyre. Comparative sequence analysis of 16S ribosomal RNA (rRNA) gene libraries revealed high diversity and significant spatial variability within the class Flavobacteria. Published and newly designed oligonucleotide probes were used to enumerate eleven flavobacterial clades by catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH). We found that different provinces harbor distinct flavobacterial communities. Clade DE2 accounted for a substantial fraction of total Flavobacteria only in the Polar Biome (BPLR), whereas the VISION clades VIS1 and VIS4 significantly increased in the Arctic (ARCT) province. Members of the genus Polaribacter were the most abundant clade in all the water masses analyzed, with highest absolute numbers in BPLR and ARCT. We improved the CARD-FISH protocol to quantify the rare clades VIS2, VIS3, VIS5 and VIS6, which were present in abundances below 0.5%. They all showed pronounced regional distribution patterns. Microscopic analysis proved a specific enrichment of Flavobacteria in the phycosphere of nanophytoplankton of BPLR and ARCT. Our results suggest that different marine flavobacterial clades have distinct niches and different life strategies.