Acidimicrobiia, the actinomycetota of coastal marine sediments: Abundance, taxonomy and genomic potential.

Microbial communities in marine sediments represent some of the densest and most diverse biological communities known, with up to a billion cells and thousands of species per milliliter. Among this taxonomic diversity, the class Acidimicrobiia, within the phylum Actinomycetota, stands out for its consistent presence, yet its limited taxonomic understanding obscures its ecological role. We used metagenome-assembled genomes from a 5-year Arctic fjord sampling campaign and compared them to publicly available Acidimicrobiia genomes using 16S rRNA gene and whole-genome phylogenies, alongside gene prediction and annotation to study their taxonomy and genomic potential. Overall, we provide a taxonomic overview of the class Acidimicrobiia and show its significant prevalence in Isfjorden and Helgoland coastal sediments, representing over 90% of Actinomycetota 16S rRNA gene sequences, and 3-7% of Bacteria. We propose Benthobacter isfjordensis gen. nov., sp. nov., Hadalibacter litoralis gen. nov., sp. nov., and two new species from Ilumatobacter, following SeqCode guidelines. In addition, we report the first in situ quantification of the family Ilumatobacteraceae, revealing its substantial presence (1-6%) in coastal sediments. This work highlights the need of refining the taxonomy of Acidimicrobiia to better understand their ecological contributions.

Distinct actors drive different mechanisms of biopolymer processing in polar marine coastal sediments.

Heterotrophic bacteria in the ocean initiate biopolymer degradation using extracellular enzymes that yield low molecular weight hydrolysis products in the environment, or by using a selfish uptake mechanism that retains the hydrolysate for the enzyme-producing cell. The mechanism used affects the availability of hydrolysis products to other bacteria, and thus also potentially the composition and activity of the community. In marine systems, these two mechanisms of substrate processing have been studied in the water column, but to date, have not been investigated in sediments. In surface sediments from an Arctic fjord of Svalbard, we investigated mechanisms of biopolymer hydrolysis using four polysaccharides and mucin, a glycoprotein. Extracellular hydrolysis of all biopolymers was rapid. Moreover, rapid degradation of mucin suggests that it may be a key substrate for benthic microbes. Although selfish uptake is common in ocean waters, only a small fraction (0.5%-2%) of microbes adhering to sediments used this mechanism. Selfish uptake was carried out primarily by Planctomycetota and Verrucomicrobiota. The overall dominance of extracellular hydrolysis in sediments, however, suggests that the bulk of biopolymer processing is carried out by a benthic community relying on the sharing of enzymatic capabilities and scavenging of public goods.

Genes for laminarin degradation are dispersed in the genomes of particle-associated Maribacter species.

Laminarin is a cytosolic storage polysaccharide of phytoplankton and macroalgae and accounts for over 10% of the world's annually fixed carbon dioxide. Algal disruption, for example, by viral lysis releases laminarin. The soluble sugar is rapidly utilized by free-living planktonic bacteria, in which sugar transporters and the degrading enzymes are frequently encoded in polysaccharide utilization loci. The annotation of flavobacterial genomes failed to identify canonical laminarin utilization loci in several particle-associated bacteria, in particular in strains of Maribacter. In this study, we report in vivo utilization of laminarin by Maribacter forsetii accompanied by additional cell growth and proliferation. Laminarin utilization coincided with the induction of an extracellular endo-laminarinase, SusC/D outer membrane oligosaccharide transporters, and a periplasmic glycosyl hydrolase family 3 protein. An ABC transport system and sugar kinases were expressed. Endo-laminarinase activity was also observed in Maribacter sp. MAR_2009_72, Maribacter sp. Hel_I_7, and Maribacter dokdonensis MAR_2009_60. Maribacter dokdonensis MAR_2009_71 lacked the large endo-laminarinase gene in the genome and had no endo-laminarinase activity. In all genomes, genes of induced proteins were scattered across the genome rather than clustered in a laminarin utilization locus. These observations revealed that the Maribacter strains investigated in this study participate in laminarin utilization, but in contrast to many free-living bacteria, there is no co-localization of genes encoding the enzymatic machinery for laminarin utilization.

Niche separation in bacterial communities and activities in porewater, loosely attached, and firmly attached fractions in permeable surface sediments.

Heterotrophic microbes are central to organic matter degradation and transformation in marine sediments. Currently, most investigations of benthic microbiomes do not differentiate between processes in the porewater and on the grains and, hence, only show a generalized picture of the community. This limits our understanding of the structure and functions of sediment microbiomes. To address this problem, we fractionated sandy surface sediment microbial communities from a coastal site in Isfjorden, Svalbard, into cells associated with the porewater, loosely attached to grains, and firmly attached to grains; we found dissimilar bacterial communities and metabolic activities in these fractions. Most (84%-89%) of the cells were firmly attached, and this fraction comprised more anaerobes, such as sulfate reducers, than the other fractions. The porewater and loosely attached fractions (3% and 8%-13% of cells, respectively) had more aerobic heterotrophs. These two fractions generally showed a higher frequency of dividing cells, polysaccharide (laminarin) hydrolysis rates, and per-cell O2 consumption than the firmly attached cells. Thus, the different fractions occupy distinct niches within surface sediments: the firmly attached fraction is potentially made of cells colonizing areas on the grain that are protected from abrasion, but might be more diffusion-limited for organic matter and electron acceptors. In contrast, the porewater and loosely attached fractions are less resource-limited and have faster growth. Their cell numbers are kept low possibly through abrasion and exposure to grazers. Differences in community composition and activity of these cell fractions point to their distinct roles and contributions to carbon cycling within surface sediments.

Bacterial chemolithoautotrophy in ultramafic plumes along the Mid-Atlantic Ridge.

Hydrothermal vent systems release reduced chemical compounds that act as an important energy source in the deep sea. Chemolithoautotrophic microbes inhabiting hydrothermal plumes oxidize these compounds, in particular, hydrogen and reduced sulfur, to obtain the energy required for CO2 fixation. Here, we analysed the planktonic communities of four hydrothermal systems located along the Mid-Atlantic Ridge: Irinovskoe, Semenov-2, Logatchev-1, and Ashadze-2, by combining long-read 16S rRNA gene analysis, fluorescence in situ hybridization, meta-omics, and thermodynamic calculations. Sulfurimonas and SUP05 dominated the microbial communities in these hydrothermal plumes. Investigation of Sulfurimonas and SUP05 MAGs, and their gene transcription in plumes indicated a niche partitioning driven by hydrogen and sulfur. In addition to sulfur and hydrogen oxidation, a novel SAR202 clade inhabiting the plume, here referred to as genus Carboxydicoccus, harbours the capability for CO oxidation and CO2 fixation via reverse TCA cycle. Both pathways were also highly transcribed in other hydrogen-rich plumes, including the Von Damm vent field. Carboxydicoccus profundi reached up to 4% relative abundance (1.0 x 103 cell ml- 1) in Irinovskoe non-buoyant plume and was also abundant in non-hydrothermally influenced deep-sea metagenomes (up to 5 RPKM). Therefore, CO, which is probably not sourced from the hydrothermal fluids (1.9-5.8 µM), but rather from biological activities within the rising fluid, may serve as a significant energy source in hydrothermal plumes. Taken together, this study sheds light on the chemolithoautotrophic potential of the bacterial community in Mid-Atlantic Ridge plumes.

Polymicrobial consortia in the pathogenesis of biofilm vaginosis visualized by FISH. Historic review outlining the basic principles of the polymicrobial infection theory.

The manuscript disputes the exclusive mono-infectious way of thinking, which presumes that for every infection only one pathogen is responsible and sufficient, when infectious vectors, close contact and reduced immunity meet. In situations involving heavily colonized anatomical sites such an approach often ends in insoluble contradictions. Upon critical reflection and evaluation of 20 years research on spatial organization of vaginal microbiota it is apparent, that in some situations, pathogens may act and operate in permanent, structurally organized consortia, whereas its individual components may be innocuous and innocent, failing to express any pathogenic effect. In these cases, consortia are the true pathogens responsible for many infectious conditions, which usually remain unrecognized as long as improperly diagnosed. The structure of such consortia can be unraveled using ribosomal fluorescence in situ hybridization (FISH). FISH methodology, that not only offers an ex vivo opportunity to recognize bacterial species, but provides unique physical insight into their specific role in the pathogenesis of polymicrobial infections. Ribosomal FISH technique applied to both, women with bacterial vaginosis (BV) and their male partners, has added significantly to our understanding of the pathogenesis of this condition and contributed to appreciating the mechanisms of polymicrobial, community-based infection, potentially leading to therapeutic advances.

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

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

Proteomic insight into arabinogalactan utilization by particle-associated Maribacter sp. MAR_2009_72.

Arabinose and galactose are major, rapidly metabolized components of marine particulate and dissolved organic matter. In this study, we observed for the first time large microbiomes for the degradation of arabinogalactan and report a detailed investigation of arabinogalactan utilization by the flavobacterium Maribacter sp. MAR_2009_72. Cellular extracts hydrolysed arabinogalactan in vitro. Comparative proteomic analyses of cells grown on arabinogalactan, arabinose, galactose, and glucose revealed the expression of specific proteins in the presence of arabinogalactan, mainly glycoside hydrolases (GH). Extracellular glycan hydrolysis involved five alpha-l-arabinofuranosidases affiliating with glycoside hydrolase families 43 and 51, four unsaturated rhamnogalacturonylhydrolases (GH105) and a protein with a glycoside hydrolase family-like domain. We detected expression of three induced TonB-dependent SusC/D transporter systems, one SusC, and nine glycoside hydrolases with a predicted periplasmatic location. These are affiliated with the families GH3, GH10, GH29, GH31, GH67, GH78, and GH115. The genes are located outside of and within canonical polysaccharide utilization loci classified as specific for arabinogalactan, for galactose-containing glycans, and for arabinose-containing glycans. The breadth of enzymatic functions expressed in Maribacter sp. MAR_2009_72 as response to arabinogalactan from the terrestrial plant larch suggests that Flavobacteriia are main catalysts of the rapid turnover of arabinogalactans in the marine environment.

Particle-attached bacteria act as gatekeepers in the decomposition of complex phytoplankton polysaccharides.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: Particle-attached bacteria represented only 1% of all bloom-associated bacteria, yet our data suggest that many abundant active clades played a pivotal gatekeeping role in the solubilization and subsequent degradation of numerous important classes of algal glycans. The high diversity of polysaccharide niches among the most active particle-attached clades therefore is a determining factor for the proportion of algal polysaccharides that can be rapidly remineralized during generally short-lived phytoplankton bloom events. Video Abstract.

Taxonomic and functional stability overrules seasonality in polar benthic microbiomes.

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

Pulsed inputs of high molecular weight organic matter shift the mechanisms of substrate utilisation in marine bacterial communities.

Heterotrophic bacteria hydrolyze high molecular weight (HMW) organic matter extracellularly prior to uptake, resulting in diffusive loss of hydrolysis products. An alternative 'selfish' uptake mechanism that minimises this loss has recently been found to be common in the ocean. We investigated how HMW organic matter addition affects these two processing mechanisms in surface and bottom waters at three stations in the North Atlantic Ocean. A pulse of HMW organic matter increased cell numbers, as well as the rate and spectrum of extracellular enzymatic activities at both depths. The effects on selfish uptake were more differentiated: in Gulf Stream surface waters and productive surface waters south of Newfoundland, selfish uptake of structurally simple polysaccharides increased upon HMW organic matter addition. The number of selfish bacteria taking up structurally complex polysaccharides, however, was largely unchanged. In contrast, in the oligotrophic North Atlantic gyre, despite high external hydrolysis rates, the number of selfish bacteria was unchanged, irrespective of polysaccharide structure. In deep bottom waters (> 4000 m), structurally complex substrates were processed only by selfish bacteria. Mechanisms of substrate processing-and the extent to which hydrolysis products are released to the external environment-depend on substrate structural complexity and the resident bacterial community.

Towards estimating the number of strains that make up a natural bacterial population.

What a strain is and how many strains make up a natural bacterial population remain elusive concepts despite their apparent importance for assessing the role of intra-population diversity in disease emergence or response to environmental perturbations. To advance these concepts, we sequenced 138 randomly selected Salinibacter ruber isolates from two solar salterns and assessed these genomes against companion short-read metagenomes from the same samples. The distribution of genome-aggregate average nucleotide identity (ANI) values among these isolates revealed a bimodal distribution, with four-fold lower occurrence of values between 99.2% and 99.8% relative to ANI >99.8% or <99.2%, revealing a natural "gap" in the sequence space within species. Accordingly, we used this ANI gap to define genomovars and a higher ANI value of >99.99% and shared gene-content >99.0% to define strains. Using these thresholds and extrapolating from how many metagenomic reads each genomovar uniquely recruited, we estimated that -although our 138 isolates represented about 80% of the Sal. ruber population- the total population in one saltern pond is composed of 5,500 to 11,000 genomovars, the great majority of which appear to be rare in-situ. These data also revealed that the most frequently recovered isolate in lab media was often not the most abundant genomovar in-situ, suggesting that cultivation biases are significant, even in cases that cultivation procedures are thought to be robust. The methodology and ANI thresholds outlined here should represent a useful guide for future microdiversity surveys of additional microbial species.

An ANI gap within bacterial species that advances the definitions of intra-species units.

IMPORTANCE: Bacterial strains and clonal complexes are two cornerstone concepts for microbiology that remain loosely defined, which confuses communication and research. Here we identify a natural gap in genome sequence comparisons among isolate genomes of all well-sequenced species that has gone unnoticed so far and could be used to more accurately and precisely define these and related concepts compared to current methods. These findings advance the molecular toolbox for accurately delineating and following the important units of diversity within prokaryotic species and thus should greatly facilitate future epidemiological and micro-diversity studies across clinical and environmental settings.

Carbohydrates and carbohydrate degradation gene abundance and transcription in Atlantic waters of the Arctic.

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 potential carbohydrate utilisation by microbes in Atlantic waters of the Arctic. 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, while the more labile ß-1,3-glucan exhibited a patchy distribution. Through 'omics analysis, we identify variations in the abundance and transcription of carbohydrate degradation-related genes across samples at the community and population level. The populations contributing the most to transcription were taxonomically related to those known as primary responders and key carbohydrate degraders in temperate ecosystems, such as NS4 Marine Group and Formosa. The unique transcription profiles for these populations suggest distinct substrate utilisation potentials, with predicted glycan targets corresponding to those structurally identified in POM from the same sampling sites. By combining cutting-edge technologies and protocols, we provide insights into the carbohydrate component of the carbon cycle in the Arctic during late summer and present a high-quality dataset that will be of great value for future comparative analyses.

Metagenome-assembled genomes reveal greatly expanded taxonomic and functional diversification of the abundant marine Roseobacter RCA cluster.

BACKGROUND: The RCA (Roseobacter clade affiliated) cluster belongs to the family Roseobacteracea and represents a major Roseobacter lineage in temperate to polar oceans. Despite its prevalence and abundance, only a few genomes and one described species, Planktomarina temperata, exist. To gain more insights into our limited understanding of this cluster and its taxonomic and functional diversity and biogeography, we screened metagenomic datasets from the global oceans and reconstructed metagenome-assembled genomes (MAG) affiliated to this cluster. RESULTS: The total of 82 MAGs, plus five genomes of isolates, reveal an unexpected diversity and novel insights into the genomic features, the functional diversity, and greatly refined biogeographic patterns of the RCA cluster. This cluster is subdivided into three genera: Planktomarina, Pseudoplanktomarina, and the most deeply branching Candidatus Paraplanktomarina. Six of the eight Planktomarina species have larger genome sizes (2.44-3.12 Mbp) and higher G + C contents (46.36-53.70%) than the four Pseudoplanktomarina species (2.26-2.72 Mbp, 42.22-43.72 G + C%). Cand. Paraplanktomarina is represented only by one species with a genome size of 2.40 Mbp and a G + C content of 45.85%. Three novel species of the genera Planktomarina and Pseudoplanktomarina are validly described according to the SeqCode nomenclature for prokaryotic genomes. Aerobic anoxygenic photosynthesis (AAP) is encoded in three Planktomarina species. Unexpectedly, proteorhodopsin (PR) is encoded in the other Planktomarina and all Pseudoplanktomarina species, suggesting that this light-driven proton pump is the most important mode of acquiring complementary energy of the RCA cluster. The Pseudoplanktomarina species exhibit differences in functional traits compared to Planktomarina species and adaptations to more resource-limited conditions. An assessment of the global biogeography of the different species greatly expands the range of occurrence and shows that the different species exhibit distinct biogeographic patterns. They partially reflect the genomic features of the species. CONCLUSIONS: Our detailed MAG-based analyses shed new light on the diversification, environmental adaptation, and global biogeography of a major lineage of pelagic bacteria. The taxonomic delineation and validation by the SeqCode nomenclature of prominent genera and species of the RCA cluster may be a promising way for a refined taxonomic identification of major prokaryotic lineages and sublineages in marine and other prokaryotic communities assessed by metagenomics approaches. Video Abstract.

Atlantic water influx and sea-ice cover drive taxonomic and functional shifts in Arctic marine bacterial communities.

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.

Epiphytic common core bacteria in the microbiomes of co-located green (Ulva), brown (Saccharina) and red (Grateloupia, Gelidium) macroalgae.

BACKGROUND: Macroalgal epiphytic microbial communities constitute a rich resource for novel enzymes and compounds, but studies so far largely focused on tag-based microbial diversity analyses or limited metagenome sequencing of single macroalgal species. RESULTS: We sampled epiphytic bacteria from specimens of Ulva sp. (green algae), Saccharina sp. (brown algae), Grateloupia sp. and Gelidium sp. (both red algae) together with seawater and sediment controls from a coastal reef in Weihai, China, during all seasons. Using 16S rRNA amplicon sequencing, we identified 14 core genera (consistently present on all macroalgae), and 14 dominant genera (consistently present on three of the macroalgae). Core genera represented ~ 0.7% of all genera, yet accounted for on average 51.1% of the bacterial abundances. Plate cultivation from all samples yielded 5,527 strains (macroalgae: 4,426) representing 1,235 species (685 potentially novel). Sequencing of selected strains yielded 820 non-redundant draft genomes (506 potentially novel), and sequencing of 23 sampled metagenomes yielded 1,619 metagenome-assembled genomes (MAGs), representing further 1,183 non-redundant genomes. 230 isolates and 153 genomes were obtained from the 28 core/dominant genera. We analyzed the genomic potential of phycosphere bacteria to degrade algal polysaccharides and to produce bioactive secondary metabolites. We predicted 4,451 polysaccharide utilization loci (PULs) and 8,810 biosynthetic gene clusters (BGCs). These were particularly prevalent in core/dominant genera. CONCLUSIONS: Our metabolic annotations and analyses of MAGs and genomes provide new insights into novel species of phycosphere bacteria and their ecological niches for an improved understanding of the macroalgal phycosphere microbiome. Video Abstract.

Comparing genomes recovered from time-series metagenomes using long- and short-read sequencing technologies.

BACKGROUND: Over the past years, sequencing technologies have expanded our ability to examine novel microbial metabolisms and diversity previously obscured by isolation approaches. Long-read sequencing promises to revolutionize the metagenomic field and recover less fragmented genomes from environmental samples. Nonetheless, how to best benefit from long-read sequencing and whether long-read sequencing can provide recovered genomes of similar characteristics as short-read approaches remains unclear. RESULTS: We recovered metagenome-assembled genomes (MAGs) from the free-living fraction at four-time points during a spring bloom in the North Sea. The taxonomic composition of all MAGs recovered was comparable between technologies. However, differences consisted of higher sequencing depth for contigs and higher genome population diversity in short-read compared to long-read metagenomes. When pairing population genomes recovered from both sequencing approaches that shared ≥ 99% average nucleotide identity, long-read MAGs were composed of fewer contigs, a higher N50, and a higher number of predicted genes when compared to short-read MAGs. Moreover, 88% of the total long-read MAGs carried a 16S rRNA gene compared to only 23% of MAGs recovered from short-read metagenomes. Relative abundances for population genomes recovered using both technologies were similar, although disagreements were observed for high and low GC content MAGs. CONCLUSIONS: Our results highlight that short-read technologies recovered more MAGs and a higher number of species than long-read due to an overall higher sequencing depth. Long-read samples produced higher quality MAGs and similar species composition compared to short-read sequencing. Differences in the GC content recovered by each sequencing technology resulted in divergences in the diversity recovered and relative abundance of MAGs within the GC content boundaries.

In situ cell division and mortality rates of SAR11, SAR86, Bacteroidetes, and Aurantivirga during phytoplankton blooms reveal differences in population controls.

Net growth of microbial populations, that is, changes in abundances over time, can be studied using 16S rRNA fluorescence in situ hybridization (FISH). However, this approach does not differentiate between mortality and cell division rates. We used FISH-based image cytometry in combination with dilution culture experiments to study net growth, cell division, and mortality rates of four bacterial taxa over two distinct phytoplankton blooms: the oligotrophs SAR11 and SAR86, and the copiotrophic phylum Bacteroidetes, and its genus Aurantivirga. Cell volumes, ribosome content, and frequency of dividing cells (FDC) co-varied over time. Among the three, FDC was the most suitable predictor to calculate cell division rates for the selected taxa. The FDC-derived cell division rates for SAR86 of up to 0.8/day and Aurantivirga of up to 1.9/day differed, as expected for oligotrophs and copiotrophs. Surprisingly, SAR11 also reached high cell division rates of up to 1.9/day, even before the onset of phytoplankton blooms. For all four taxonomic groups, the abundance-derived net growth (-0.6 to 0.5/day) was about an order of magnitude lower than the cell division rates. Consequently, mortality rates were comparably high to cell division rates, indicating that about 90% of bacterial production is recycled without apparent time lag within 1 day. Our study shows that determining taxon-specific cell division rates complements omics-based tools and provides unprecedented clues on individual bacterial growth strategies including bottom-up and top-down controls. IMPORTANCE The growth of a microbial population is often calculated from their numerical abundance over time. However, this does not take cell division and mortality rates into account, which are important for deriving ecological processes like bottom-up and top-down control. In this study, we determined growth by numerical abundance and calibrated microscopy-based methods to determine the frequency of dividing cells and subsequently calculate taxon-specific cell division rates in situ. The cell division and mortality rates of two oligotrophic (SAR11 and SAR86) and two copiotrophic (Bacteroidetes and Aurantivirga) taxa during two spring phytoplankton blooms showed a tight coupling for all four taxa throughout the blooms without any temporal offset. Unexpectedly, SAR11 showed high cell division rates days before the bloom while cell abundances remained constant, which is indicative of strong top-down control. Microscopy remains the method of choice to understand ecological processes like top-down and bottom-up control on a cellular level.