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Viruses play a pivotal role in ecosystems by influencing biochemical cycles and impacting the structure and evolution of their host cells. The widespread pelagiphages infect Pelagibacter spp., the most abundant marine microbe on Earth, and thus play a significant role in carbon transformation through the viral shunt. Among these viruses, the uncultured lytic pelagiphage vSAG 37-F6, uncovered by single-virus genomics, is likely the most numerous virus in the ocean. While previous research has delved into the diversity and spatial distribution of vSAG 37-F6, there is still a gap in understanding its temporal dynamics, hindering our insight into its ecological impact. We explored the temporal dynamics of vSAG 37-F6, assessing periodic fluctuations in abundance and evolutionary patterns using long- and short-term data series. In the long-term series (7 years), metagenomics showed negative selection acting on all viral genes, with a highly conserved overall diversity over time composed of a pool of yearly emergent, highly similar novel strains that exhibited a seasonal abundance pattern with two peaks during winter and fall and a decrease in months with higher UV radiation. Most non-synonymous polymorphisms occurred in structural viral proteins located in regions with low conformational restrictions, suggesting that many of the viral genes of this population are highly purified over its evolution. At the fine-scale resolution (24 h time series), combining digital PCR and metagenomics, we identified two peaks of cellular infection for the targeted vSAG 37-F6 viral strain (up to approximately 10 3 copies/ng of prokaryotic DNA), one before sunrise and the second shortly after midday. Considering the high number of co-occurring strains of this microdiverse virus, the abundance values at the species or genus level could be orders of magnitudes higher. These findings represent a significant advancement in understanding the dynamics of the potentially most abundant oceanic virus, providing valuable insights into ecologically relevant marine viruses.

Prokaryotic abundance and activity are commonly assessed by dividing them into two size-fractions: free-living and attached to particles. Nevertheless, organic matter, essential for the growth of heterotrophic prokaryotes, is present in the environment in a continuum of sizes, from purely dissolved to large particles. Therefore, defining the activity of the prokaryotic community would be more accurate by considering all the distinct size fractions. To achieve this, we measured prokaryotic abundance (PA), heterotrophic prokaryotic activity (as leucine incorporation) and extracellular enzyme activities at a coastal site in the NW Mediterranean Sea. We conducted measurements in both bulk seawater and size fractionated samples sequentially passing through 5 different filter types: 0.2-0.8-3-5-10 μm pore size. Our results indicate that the fraction <0.8 μm contained the highest percentage of cells (91.6 ± 1.1 %) and leucine incorporation rates (72.2 ± 3.5 %). Most of the extracellular enzyme activity appeared in the dissolved fraction (<0.2 μm; 19.8-79.4 %), yet the specific activity of the enzymes (per cell activity) was 100-1000 times higher in the particulate (>0.8 μm) than in the free-living (0.2-0.8 μm) fraction. The size fraction with highest specific activities for leucine incorporation and most of the enzyme activities (β-glucosidase, esterase, Leu-aminopeptidase and alkaline phosphatase) was the 5-10 μm fraction. In contrast, the higher specific chitobiase activity in the >10 μm fraction, suggests that the prokaryotic community colonizing large particles might be more specialized in the hydrolysis of organic matter of zooplanktonic origin than the community colonizing smaller particles., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Marine microbial communities differ genetically, metabolically, and ecologically according to their lifestyle, and they may respond differently to environmental changes. In this study, we investigated the seasonal dynamics of bacterial assemblies in the free-living (FL) and particle-associated (PA) fractions across a span of 6 years in the Blanes Bay Microbial Observatory in the Northwestern Mediterranean. Both lifestyles showed marked seasonality. The trends in alpha diversity were similar, with lower values in spring-summer than in autumn-winter. Samples from both fractions were grouped seasonally and the percentage of community variability explained by the measured environmental variables was comparable (32% in FL and 31% in PA). Canonical analyses showed that biotic interactions were determinants of bacterioplankton dynamics and that their relevance varies depending on lifestyles. Time-decay curves confirmed a high degree of predictability in both fractions. Yet, 'seasonal' Amplicon Sequence Variants (ASVs) (as defined by Lomb Scargle time series analysis) in the PA communities represented 46% of the total relative abundance while these accounted for 30% in the FL fraction. These results demonstrate that bacteria inhabiting both fractions exhibit marked seasonality, highlighting the importance of accounting for both lifestyles to fully comprehend the dynamics of marine prokaryotic communities., (© 2024 The Author(s). Environmental Microbiology Reports published by John Wiley & Sons Ltd.)

COVID-19 has led to global population lockdowns that have had indirect effects on terrestrial and marine fauna, yet little is known on their effects on marine planktonic communities. We analysed the effect of the spring 2020 lockdown in a marine coastal area in Blanes Bay, NW Mediterranean. We compared a set of 23 oceanographic, microbial and biogeochemical variables sampled right after the strict lockdown in Spain, with data from the previous 15 years after correcting for long-term trends. Our analysis shows a series of changes in the microbial communities which may have been induced by the combination of the decreased nitrogen atmospheric load, the lower wastewater flux and the reduced fishing activity in the area, among other factors. In particular, we detected a slight decrease beyond the long-term trend in chlorophyll a, in the abundance of several microbial groups (phototrophic nanoflagellates and total prokaryotes) and in prokaryotic activity (heterotrophic prokaryotic production and β-glucosidase activity) which, as a whole, resulted in a moderate increase of oligotrophy in Blanes Bay after the lockdown., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The deep sea, the largest ocean's compartment, drives planetary-scale biogeochemical cycling. Yet, the functional exploration of its microbial communities lags far behind other environments. Here we analyze 58 metagenomes from tropical and subtropical deep oceans to generate the Malaspina Gene Database. Free-living or particle-attached lifestyles drive functional differences in bathypelagic prokaryotic communities, regardless of their biogeography. Ammonia and CO oxidation pathways are enriched in the free-living microbial communities and dissimilatory nitrate reduction to ammonium and H 2 oxidation pathways in the particle-attached, while the Calvin Benson-Bassham cycle is the most prevalent inorganic carbon fixation pathway in both size fractions. Reconstruction of the Malaspina Deep Metagenome-Assembled Genomes reveals unique non-cyanobacterial diazotrophic bacteria and chemolithoautotrophic prokaryotes. The widespread potential to grow both autotrophically and heterotrophically suggests that mixotrophy is an ecologically relevant trait in the deep ocean. These results expand our understanding of the functional microbial structure and metabolic capabilities of the largest Earth aquatic ecosystem.

Deep ocean microbial communities rely on the organic carbon produced in the sunlit ocean, yet it remains unknown whether surface processes determine the assembly and function of bathypelagic prokaryotes to a larger extent than deep-sea physicochemical conditions. Here, we explored whether variations in surface phytoplankton assemblages across Atlantic, Pacific and Indian ocean stations can explain structural changes in bathypelagic (ca. 4,000 m) free-living and particle-attached prokaryotic communities (characterized through 16S rRNA gene sequencing), as well as changes in prokaryotic activity and dissolved organic matter (DOM) quality. We show that the spatial structuring of prokaryotic communities in the bathypelagic strongly followed variations in the abundances of surface dinoflagellates and ciliates, as well as gradients in surface primary productivity, but were less influenced by bathypelagic physicochemical conditions. Amino acid-like DOM components in the bathypelagic reflected variations of those components in surface waters, and seemed to control bathypelagic prokaryotic activity. The imprint of surface conditions was more evident in bathypelagic than in shallower mesopelagic (200-1,000 m) communities, suggesting a direct connectivity through fast-sinking particles that escape mesopelagic transformations. Finally, we identified a pool of endemic deep-sea prokaryotic taxa (including potentially chemoautotrophic groups) that appear less connected to surface processes than those bathypelagic taxa with a widespread vertical distribution. Our results suggest that surface planktonic communities shape the spatial structure of the bathypelagic microbiome to a larger extent than the local physicochemical environment, likely through determining the nature of the sinking particles and the associated prokaryotes reaching bathypelagic waters., (© 2020 John Wiley & Sons Ltd.)

Recent developments in community and single-cell genomic approaches have provided an unprecedented amount of information on the ecology of microbes in the aquatic environment. However, linkages between each specific microbe's identity and their in situ level of activity (be it growth, division or just metabolic activity) are much more scarce. The ultimate goal of marine microbial ecology is to understand how the environment determines the types of different microbes in nature, their function, morphology and cell-to-cell interactions and to do so we should gather three levels of information, the genomic (including identity), the functional (activity or growth), and the morphological, and for as many individual cells as possible. We present a brief overview of methodologies applied to address single-cell activity in marine prokaryotes, together with a discussion of the difficulties in identifying and categorizing activity and growth. We then provide and discuss some examples showing how visualization has been pivotal for challenging established paradigms and for understanding the role of microbes in the environment, unveiling processes and interactions that otherwise would have been overlooked. We conclude by stating that more effort should be directed towards integrating visualization in future approaches if we want to gain a comprehensive insight into how microbes contribute to the functioning of ecosystems. This article is part of a discussion meeting issue 'Single cell ecology'.

The ocean is home to myriad small planktonic organisms that underpin the functioning of marine ecosystems. However, their spatial patterns of diversity and the underlying drivers remain poorly known, precluding projections of their responses to global changes. Here we investigate the latitudinal gradients and global predictors of plankton diversity across archaea, bacteria, eukaryotes, and major virus clades using both molecular and imaging data from Tara Oceans. We show a decline of diversity for most planktonic groups toward the poles, mainly driven by decreasing ocean temperatures. Projections into the future suggest that severe warming of the surface ocean by the end of the 21 st century could lead to tropicalization of the diversity of most planktonic groups in temperate and polar regions. These changes may have multiple consequences for marine ecosystem functioning and services and are expected to be particularly significant in key areas for carbon sequestration, fisheries, and marine conservation. VIDEO ABSTRACT., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

We studied the long-term temporal dynamics of the aerobic anoxygenic phototrophic (AAP) bacteria, a relevant functional group in the coastal marine microbial food web, using high-throughput sequencing of the pufM gene coupled with multivariate, time series and co-occurrence analyses at the Blanes Bay Microbial Observatory (NW Mediterranean). Additionally, using metagenomics, we tested whether the used primers captured accurately the seasonality of the most relevant AAP groups. Phylogroup K (Gammaproteobacteria) was the greatest contributor to community structure over all seasons, with phylogroups E and G (Alphaproteobacteria) being prevalent in spring. Diversity indices showed a clear seasonal trend, with maximum values in winter, which was inverse to that of AAP abundance. Multivariate analyses revealed sample clustering by season, with a relevant proportion of the variance explained by day length, temperature, salinity, phototrophic nanoflagellate abundance, chlorophyll a, and silicate concentration. Time series analysis showed robust rhythmic patterns of co-occurrence, but distinct seasonal behaviors within the same phylogroup, and even within different amplicon sequence variants (ASVs) conforming the same operational taxonomic unit (OTU). Altogether, our results picture the AAP assemblage as highly seasonal and recurrent but containing ecotypes showing distinctive temporal niche partitioning, rather than being a cohesive functional group.

Microbial taxa range from being ubiquitous and abundant across space to extremely rare and endemic, depending on their ecophysiology and on different processes acting locally or regionally. However, little is known about how cosmopolitan or rare taxa combine to constitute communities and whether environmental variations promote changes in their relative abundances. Here we identified the Spatial Abundance Distribution (SpAD) of individual prokaryotic taxa (16S rDNA-defined Operational Taxonomic Units, OTUs) across 108 globally-distributed surface ocean stations. We grouped taxa based on their SpAD shape ("normal-like"- abundant and ubiquitous; "logistic"- globally rare, present in few sites; and "bimodal"- abundant only in certain oceanic regions), and investigated how the abundance of these three categories relates to environmental gradients. Most surface assemblages were numerically dominated by a few cosmopolitan "normal-like" OTUs, yet there was a gradual shift towards assemblages dominated by "logistic" taxa in specific areas with productivity and temperature differing the most from the average conditions in the sampled stations. When we performed the SpAD categorization including additional habitats (deeper layers and particles of varying sizes), the SpAD of many OTUs changed towards fewer "normal-like" shapes, and OTUs categorized as globally rare in the surface ocean became abundant. This suggests that understanding the mechanisms behind microbial rarity and dominance requires expanding the context of study beyond local communities and single habitats. We show that marine bacterial communities comprise taxa displaying a continuum of SpADs, and that variations in their abundances can be linked to habitat transitions or barriers that delimit the distribution of community members., (© 2019 John Wiley & Sons Ltd.)

The impact of grazing, resource competition and light on prokaryotic growth and taxonomic composition in subtropical and tropical surface waters were studied through 10 microcosm experiments conducted between 30°N and 30°S in the Atlantic, Pacific and Indian oceans. Under natural sunlight conditions, significant changes in taxonomic composition were only observed after the reduction of grazing by sample filtration in combination with a decrease in resource competition by sample dilution. Sunlight exposure significantly reduced prokaryote growth (11 ± 6%) and community richness (14 ± 4%) compared to continuous darkness but did not significantly change community composition. The largest growth inhibition after sunlight exposure occurred at locations showing deep mixed layers. The reduction of grazing had an expected and significant positive effect on growth, but caused a significant decrease in community richness (16 ± 6%), suggesting that the coexistence of many different OTUs is partly promoted by the presence of predators. Dilution of the grazer-free prokaryotic community significantly enhanced growth at the level of community, but consistently and sharply reduced the abundance of Prochlorococcus and SAR11 populations. The decline of these oligotrophic bacterial taxa following an increase in resource availability is consistent with their high specialization for exploiting the limited resources available in the oligotrophic warm ocean., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The anthropogenic perturbation of the phosphorus (P) marine biogeochemical cycle due to synthetic organophosphorus compounds remains unexplored. The objective of this work was to investigate the microbial degradation of organophosphate triesters (OPEs), widely used as plasticizers and flame retardants, in seawater and their effects on the physiology and composition of microbial communities. Experiments were performed in July 2014 using surface seawater from the Blanes Bay Microbial Observatory (NW Mediterranean) to which OPEs were added at environmentally relevant concentrations. The concentrations of OPEs in the dissolved-phase generally decreased after 24 hours of incubation at in situ conditions. The fitted first order reaction constants were significantly different than zero for the trihaloalkyl phosphate, tris(2-chloroethyl) phosphate and trialyl phosphate tricresyl phosphate. In general, OPEs triggered an increase of the percentage of actively respiring bacteria, total bacterial activity, and the number of low-nucleic acid bacteria, and a decrease in the percentage of membrane-compromised bacteria. Members of some bacterial groups, in particular Flavobacteria, increased their specific activity, indicating that seawater contains bacteria with the potential to degrade OPEs. In aged seawater that was presumably depleted of labile dissolved organic carbon and inorganic P, alkaline phosphatase activities significantly decreased when OPEs were added, indicating a relief on P stress, consistent with the role of OPEs as potential P sources.

The bathypelagic ocean is one of the largest ecosystems on Earth and sustains half of the ocean's microbial activity. This microbial activity strongly relies on surface-derived particles, but there is growing evidence that the carbon released through solubilization of these particles may not be sufficient to meet the energy demands of deep ocean prokaryotes. To explore how bathypelagic prokaryotes respond to the absence of external inputs of carbon, we followed the long-term (1 year) dynamics of an enclosed community. Despite the lack of external energy supply, we observed a continuous succession of active prokaryotic phylotypes, which was driven by recruitment of taxa from the seed bank (i.e., initially rare operational taxonomic units [OTUs]). A single OTU belonging to Marine Group I of Thaumarchaeota, which was originally rare, dominated the microbial community for ∼ 4 months and played a fundamental role in this succession likely by introducing new organic carbon through chemolithoautotrophy. This carbon presumably produced a priming effect, because after the decline of Thaumarchaeota, the diversity and metabolic potential of the community increased back to the levels present at the start of the experiment. Our study demonstrates the profound versatility of deep microbial communities when facing organic carbon deprivation., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Microbes drive ecosystems under constraints imposed by viruses. However, a lack of virus genome information hinders our ability to answer fundamental, biological questions concerning microbial communities. Here we apply single-virus genomics (SVGs) to assess whether portions of marine viral communities are missed by current techniques. The majority of the here-identified 44 viral single-amplified genomes (vSAGs) are more abundant in global ocean virome data sets than published metagenome-assembled viral genomes or isolates. This indicates that vSAGs likely best represent the dsDNA viral populations dominating the oceans. Species-specific recruitment patterns and virome simulation data suggest that vSAGs are highly microdiverse and that microdiversity hinders the metagenomic assembly, which could explain why their genomes have not been identified before. Altogether, SVGs enable the discovery of some of the likely most abundant and ecologically relevant marine viral species, such as vSAG 37-F6, which were overlooked by other methodologies.

Ocean microbes drive biogeochemical cycling on a global scale. However, this cycling is constrained by viruses that affect community composition, metabolic activity, and evolutionary trajectories. Owing to challenges with the sampling and cultivation of viruses, genome-level viral diversity remains poorly described and grossly understudied, with less than 1% of observed surface-ocean viruses known. Here we assemble complete genomes and large genomic fragments from both surface- and deep-ocean viruses sampled during the Tara Oceans and Malaspina research expeditions, and analyse the resulting 'global ocean virome' dataset to present a global map of abundant, double-stranded DNA viruses complete with genomic and ecological contexts. A total of 15,222 epipelagic and mesopelagic viral populations were identified, comprising 867 viral clusters (defined as approximately genus-level groups). This roughly triples the number of known ocean viral populations and doubles the number of candidate bacterial and archaeal virus genera, providing a near-complete sampling of epipelagic communities at both the population and viral-cluster level. We found that 38 of the 867 viral clusters were locally or globally abundant, together accounting for nearly half of the viral populations in any global ocean virome sample. While two-thirds of these clusters represent newly described viruses lacking any cultivated representative, most could be computationally linked to dominant, ecologically relevant microbial hosts. Moreover, we identified 243 viral-encoded auxiliary metabolic genes, of which only 95 were previously known. Deeper analyses of four of these auxiliary metabolic genes (dsrC, soxYZ, P-II (also known as glnB) and amoC) revealed that abundant viruses may directly manipulate sulfur and nitrogen cycling throughout the epipelagic ocean. This viral catalog and functional analyses provide a necessary foundation for the meaningful integration of viruses into ecosystem models where they act as key players in nutrient cycling and trophic networks.

Two mesocosms experiments were conducted in winter 2010 and summer 2011 to examine how increased pCO2 and/or nutrient concentrations potentially perturbate dissolved organic matter dynamics in natural microbial assemblages. The fluorescence signals of protein- and humic-like compounds were used as a proxy for labile and non-labile material, respectively, while the evolution of bacterial populations, chlorophyll a (Chl a) and dissolved organic carbon (DOC) concentrations were used as a proxy for biological activity. For both seasons, the presence of elevated pCO2 did not cause any significant change in the DOC dynamics (p-value<0.05). The conditions that showed the greatest changes in prokaryote abundances and Chl a content were those amended with nutrients, regardless of the change in pH. The temporal evolution of fluorophores and optical indices revealed that the degree of humification of the organic molecules and their molecular weight changed significantly in the nutrient-amended treatment. The generation of protein-like compounds was paired to increases in the prokaryote abundance, being higher in the nutrient-amended tanks than in the control. Different patterns in the magnitude and direction of the generation of humic-like molecules suggested that these changes depended on initial microbial populations and the availability of extra nutrient inputs. Based on our results, it is expected that in the future projected coastal scenarios the eutrophication processes will favor the transformations of labile and recalcitrant carbon regardless of changes in pCO2., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

In this work, we study the diversity of bathypelagic microbial eukaryotes (0.8-20 μm) in the global ocean. Seawater samples from 3000 to 4000 m depth from 27 stations in the Atlantic, Pacific and Indian Oceans were analyzed by pyrosequencing the V4 region of the 18S ribosomal DNA. The relative abundance of the most abundant operational taxonomic units agreed with the results of a parallel metagenomic analysis, suggesting limited PCR biases in the tag approach. Although rarefaction curves for single stations were seldom saturated, the global analysis of all sequences together suggested an adequate recovery of bathypelagic diversity. Community composition presented a large variability among samples, which was poorly explained by linear geographic distance. In fact, the similarity between communities was better explained by water mass composition (26% of the variability) and the ratio in cell abundance between prokaryotes and microbial eukaryotes (21%). Deep diversity appeared dominated by four taxonomic groups (Collodaria, Chrysophytes, Basidiomycota and MALV-II) appearing in different proportions in each sample. Novel diversity amounted to 1% of the pyrotags and was lower than expected. Our study represents an essential step in the investigation of bathypelagic microbial eukaryotes, indicating dominating taxonomic groups and suggesting idiosyncratic assemblages in distinct oceanic regions.

Upon phosphorus (P) deficiency, marine phytoplankton reduce their requirements for P by replacing membrane phospholipids with alternative non-phosphorus lipids. It was very recently demonstrated that a SAR11 isolate also shares this capability when phosphate starved in culture. Yet, the extent to which this process occurs in other marine heterotrophic bacteria and in the natural environment is unknown. Here, we demonstrate that the substitution of membrane phospholipids for a variety of non-phosphorus lipids is a conserved response to P deficiency among phylogenetically diverse marine heterotrophic bacteria, including members of the Alphaproteobacteria and Flavobacteria. By deletion mutagenesis and complementation in the model marine bacterium Phaeobacter sp. MED193 and heterologous expression in recombinant Escherichia coli, we confirm the roles of a phospholipase C (PlcP) and a glycosyltransferase in lipid remodelling. Analyses of the Global Ocean Sampling and Tara Oceans metagenome data sets demonstrate that PlcP is particularly abundant in areas characterized by low phosphate concentrations. Furthermore, we show that lipid remodelling occurs seasonally and responds to changing nutrient conditions in natural microbial communities from the Mediterranean Sea. Together, our results point to the key role of lipid substitution as an adaptive strategy enabling heterotrophic bacteria to thrive in the vast P-depleted areas of the ocean.

The unicellular cyanobacterium UCYN-A, one of the major contributors to nitrogen fixation in the open ocean, lives in symbiosis with single-celled phytoplankton. UCYN-A includes several closely related lineages whose partner fidelity, genome-wide expression and time of evolutionary divergence remain to be resolved. Here we detect and distinguish UCYN-A1 and UCYN-A2 lineages in symbiosis with two distinct prymnesiophyte partners in the South Atlantic Ocean. Both symbiotic systems are lineage specific and differ in the number of UCYN-A cells involved. Our analyses infer a streamlined genome expression towards nitrogen fixation in both UCYN-A lineages. Comparative genomics reveal a strong purifying selection in UCYN-A1 and UCYN-A2 with a diversification process ∼91 Myr ago, in the late Cretaceous, after the low-nutrient regime period occurred during the Jurassic. These findings suggest that UCYN-A diversified in a co-evolutionary process, wherein their prymnesiophyte partners acted as a barrier driving an allopatric speciation of extant UCYN-A lineages.

The deep-sea is the largest biome of the biosphere, and contains more than half of the whole ocean's microbes. Uncovering their general patterns of diversity and community structure at a global scale remains a great challenge, as only fragmentary information of deep-sea microbial diversity exists based on regional-scale studies. Here we report the first globally comprehensive survey of the prokaryotic communities inhabiting the bathypelagic ocean using high-throughput sequencing of the 16S rRNA gene. This work identifies the dominant prokaryotes in the pelagic deep ocean and reveals that 50% of the operational taxonomic units (OTUs) belong to previously unknown prokaryotic taxa, most of which are rare and appear in just a few samples. We show that whereas the local richness of communities is comparable to that observed in previous regional studies, the global pool of prokaryotic taxa detected is modest (~3600 OTUs), as a high proportion of OTUs are shared among samples. The water masses appear to act as clear drivers of the geographical distribution of both particle-attached and free-living prokaryotes. In addition, we show that the deep-oceanic basins in which the bathypelagic realm is divided contain different particle-attached (but not free-living) microbial communities. The combination of the aging of the water masses and a lack of complete dispersal are identified as the main drivers for this biogeographical pattern. All together, we identify the potential of the deep ocean as a reservoir of still unknown biological diversity with a higher degree of spatial complexity than hitherto considered.

The transformation of leucine incorporation rates to prokaryotic carbon production rates requires the use of either theoretical or empirically determined conversion factors. Empirical leucine-to-carbon conversion factors (eCFs) vary widely across environments, and little is known about their potential controlling factors. We conducted 10 surface seawater manipulation experiments across the world's oceans, where the growth of the natural prokaryotic assemblages was promoted by filtration (i.e., removal of grazers [F treatment]) or filtration combined with dilution (i.e., also relieving resource competition [FD treatment]). The impact of sunlight exposure was also evaluated in the FD treatments, and we did not find a significant effect on the eCFs. The eCFs varied from 0.09 to 1.47 kg C mol Leu(-1) and were significantly lower in the FD than in the F samples. Also, changes in bacterial community composition during the incubations, as assessed by automated ribosomal intergenic spacer analysis (ARISA), were more pronounced in the FD than in the F treatments, compared to unmanipulated controls. Thus, we discourage the common procedure of diluting samples (in addition to filtration) for eCF determination. The eCFs in the filtered treatment were negatively correlated with the initial chlorophyll a concentration, picocyanobacterial abundance (mostly Prochlorococcus), and the percentage of heterotrophic prokaryotes with high nucleic acid content (%HNA). The latter two variables explained 80% of the eCF variability in the F treatment, supporting the view that both Prochlorococcus and HNA prokaryotes incorporate leucine in substantial amounts, although this results in relatively low carbon production rates in the oligotrophic ocean., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Prokaryotes are extremely abundant in the ocean where they drive biogeochemical cycles. The recent development and application of -omics techniques has provided an astonishing amount of information revealing the existence of a vast diversity of functional genes and a large heterogeneity within each gene. The big challenge for microbial ecologists is now to understand the ecological relevance of this variability for ecosystem functioning, a question that remains largely understudied. This brief review highlights some of the latest advances in the study of the diversity of biogeochemically relevant functional genes in the sunlit ocean., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Microbes are dominant drivers of biogeochemical processes, yet drawing a global picture of functional diversity, microbial community structure, and their ecological determinants remains a grand challenge. We analyzed 7.2 terabases of metagenomic data from 243 Tara Oceans samples from 68 locations in epipelagic and mesopelagic waters across the globe to generate an ocean microbial reference gene catalog with >40 million nonredundant, mostly novel sequences from viruses, prokaryotes, and picoeukaryotes. Using 139 prokaryote-enriched samples, containing >35,000 species, we show vertical stratification with epipelagic community composition mostly driven by temperature rather than other environmental factors or geography. We identify ocean microbial core functionality and reveal that >73% of its abundance is shared with the human gut microbiome despite the physicochemical differences between these two ecosystems., (Copyright © 2015, American Association for the Advancement of Science.)

Viruses influence ecosystems by modulating microbial population size, diversity, metabolic outputs, and gene flow. Here, we use quantitative double-stranded DNA (dsDNA) viral-fraction metagenomes (viromes) and whole viral community morphological data sets from 43 Tara Oceans expedition samples to assess viral community patterns and structure in the upper ocean. Protein cluster cataloging defined pelagic upper-ocean viral community pan and core gene sets and suggested that this sequence space is well-sampled. Analyses of viral protein clusters, populations, and morphology revealed biogeographic patterns whereby viral communities were passively transported on oceanic currents and locally structured by environmental conditions that affect host community structure. Together, these investigations establish a global ocean dsDNA viromic data set with analyses supporting the seed-bank hypothesis to explain how oceanic viral communities maintain high local diversity., (Copyright © 2015, American Association for the Advancement of Science.)

The abundance and diversity of aerobic anoxygenic phototrophs (AAPs) were studied for a year cycle at the Blanes Bay Microbial Observatory (NW Mediterranean) and their potential links to an array of environmental variables were explored. Cell numbers were low in winter and peaked in summer, showing a marked seasonality that positively correlated with day length and light at the surface. Bacteriochlorophyll a concentration, their light-harvesting pigment, was only detected between April and October, and pigment cell quota showed large variations during this period. Pyrosequencing analysis of the pufM gene revealed that the most abundant operational taxonomic units (OTUs) were affiliated to phylogroup K (Gammaproteobacteria) and uncultured phylogroup C, although they were outnumbered by alphaproteobacterial OTUs in spring. Overall, richness was higher in winter than in summer, showing an opposite trend to abundance and day length. Clustering of samples by multivariate analyses showed a clear seasonality that suggests a succession of different AAP subpopulations over time. Temperature, chlorophyll a and day length were the environmental drivers that best explained the distribution of AAP assemblages. These results indicate that AAP bacteria are highly dynamic and undergo seasonal variations in diversity and abundance mostly dictated by environmental conditions as exemplified by light availability., (© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Microbial isolates are useful models for physiological and ecological studies and can also be used to reassemble genomes from metagenomic analyses. However, the phylogenetic diversity that can be found among cultured marine bacteria may vary significantly depending on the isolation. Therefore, this study describes a set of 136 bacterial isolates obtained by traditional isolation techniques from the Blanes Bay Microbial Observatory, of which seven strains have had the whole genome sequenced. The complete set was compared to a series of environmental sequences obtained by culture-independent techniques (60 DGGE sequences and 303 clone library sequences) previously obtained by molecular methods. In this way, each isolate was placed in both its "ecological" (time of year, nutrient limitation, chlorophyll and temperature values) context or setting, and its "phylogenetic" landscape (i.e. similar organisms that were found by culture-independent techniques, when they were relevant, and when they appeared). Nearly all isolates belonged to the Gammaproteobacteria, Alphaproteobacteria, or the Bacteroidetes (70, 40 and 20 isolates, respectively). Rarefaction analyses showed similar diversity patterns for sequences from isolates and molecular approaches, except for Alphaproteobacteria where cultivation retrieved a higher diversity per unit effort. Approximately 30% of the environmental clones and isolates formed microdiversity clusters constrained at 99% 16S rRNA gene sequence identity, but the pattern was different in Bacteroidetes (less microdiversity) than in the other main groups. Seventeen cases (12.5%) of nearly complete (98-100%) rRNA sequence identity between isolates and environmental sequences were found: nine in the Alphaproteobacteria, five in the Gammaproteobacteria, and three in the Bacteroidetes, indicating that cultivation could be used to obtain at least some organisms representative of the various taxa detected by molecular methods. Collectively, these results illustrated the largely unexplored potential of culturing on standard media for complementing the study of microbial diversity by culture-independent techniques and for obtaining phylogenetically distinct model organisms from natural seawater., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

The abundance and structure of Bacteroidetes populations at diverse temporal and spatial scales were investigated in the Northwestern Mediterranean Sea. At a temporal scale, their relative abundance exhibited a marked seasonality, since it was higher in spring and decreased in winter. Similarly, Bacteroidetes community structure encompassed three main groups (winter, spring and summer-fall), which mimicked global bacterioplankton seasonality. At the spatial scale, relative abundances were similar in all surface samples along an inshore-offshore transect, but they decreased with depth. Analysis of the community structure identified four markedly different groups mostly related to different depths. Interestingly, seasonal changes in abundance and community structure were not synchronized. Furthermore, richness was higher when Bacteroidetes were less abundant. The variability of Bacteroidetes contributions to community structure in the temporal and spatial scales was correlated with different environmental factors: day length was the most important factor at the temporal scale, and salinity at the spatial scale. The community composition in terms of phylotypes changed significantly over time and along the depth gradients, but season or depth-specific phylogenetic clusters were not identified. Delineation of coherent Bacteroidetes sub-clusters should help to uncover higher resolution patterns within Bacteroidetes, and explore associations with environmental and biological variables., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Grazing rate estimates indicate that approximately half of the bacterivory in oligotrophic oceans is due to mixotrophic flagellates (MFs). However, most estimations have considered algae as a single group. Here we aimed at opening the black-box of the phytoflagellates (PFs) <20 μm. Haptophytes, chlorophytes, cryptophytes and pigmented dinoflagellates were identified using fluorescent in situ hybridization or by standard 4',6-diamidino-2-phenylindole staining. Their fluctuations in abundance, cell size, biomass and bacterivory rates were measured through an annual cycle in an oligotrophic coastal system. On average, we were able to assign to these groups: 37% of the total pico-PFs and 65% of the nano-PFs composition. Chlorophytes were mostly picoplanktonic and they never ingested fluorescently labeled bacteria. About 50% of the PF <20 μm biomass was represented by mixotrophic algae. Pigmented dinoflagellates were the least abundant group with little impact on bacterioplankton. Cryptophytes were quantitatively important during the coldest periods and explained about 4% of total bacterivory. Haptophytes were the most important mixotrophic group: (i) they were mostly represented by cells 3-5 μm in size present year-round; (ii) cell-specific grazing rates were comparable to those of other bacterivorous non-photosynthetic organisms, regardless of the in situ nutrient availability conditions; (iii) these organisms could acquire a significant portion of their carbon by ingesting bacteria; and (iv) haptophytes explained on average 40% of the bacterivory exerted by MFs and were responsible for 9-27% of total bacterivory at this site. Our results, when considered alongside the widespread distribution of haptophytes in the ocean, indicate that they have a key role as bacterivores in marine ecosystems.

The heterotrophic bacterial community of the Eastern Mediterranean Sea is believed to be limited by phosphorus (P) availability. This observation assumes that all bacterial groups are equally limited, something that has not been hitherto examined. To test this hypothesis, we performed nutrient addition experiments and investigated the response of probe-identified groups using microautoradiography combined with catalyzed reporter deposition fluorescence in situ hybridization. Our results show contrasting responses between the bacterial groups, with Gammaproteobacteria being the group more affected by P availability. The Roseobacter clade was likely colimited by P and nitrogen (N), whereas Bacteroidetes by P, N and organic carbon (C). In contrast, SAR11 cells were active regardless of the nutrient concentration. These results indicate that there is high heterogeneity in the nutrient limitation of the different components of the bacterioplankton community.

We determined the total and dissolved extracellular enzymatic activity (EEA) of α-glucosidase and β-glucosidase (AGase and BGase), alkaline phosphatase (APase) and leucine aminopeptidase (LAPase) activities in the epi-, meso- and bathypelagic waters of the subtropical Northeast Atlantic. EEA was also determined in treatments in which bacterial EEA was inhibited by erythromycin. Additionally, EEA decay experiments were performed with surface and deep waters to determine EEA lifetimes in both water masses. The proportion of dissolved to total EEA (66-89 %, 44-88 %, 57-82 % and 86-100 % for AGase, BGase, APase and LAPase, respectively) was generally higher than the cell-associated (i.e., particulate) EEA. The percentage of dissolved to total EEA was inversely proportional to the percentage of erythromycin-inhibited to total EEA. Since erythromycin-inhibited plus dissolved EEA equaled total EEA, this tentatively suggests that cell-associated EEA in the open oceanic water column is almost exclusively of bacterial origin. The decay constants of dissolved EEA were in the range of 0.002-0.048 h(-1) depending on the type of extracellular enzyme, temperature and depth in the water column. Although dissolved EEA can have different origins, the major contribution of Bacteria to cell-associated EEA and the long life-time of dissolved EEA suggest that Bacteria-and not mesophilic Archaea-are essentially the main producers of EEA in the open subtropical Northeast Atlantic down to bathypelagic layers.

Assumptions on the matching specificity of group-specific bacterial primers may bias the interpretation of environmental microbial studies. As available sequence data continue growing, the performance of primers and probes needs to be reevaluated. Here, we present an evaluation of several commonly used and one newly designed Bacteroidetes-specific primer (CF418). First, we revised the in silico primer coverage and specificity with the current SILVA and RDP databases. We found minor differences with previous studies, which could be explained by the chosen databases, taxonomies, and matching criteria. We selected eight commonly used Bacteroidetes primers and tested them with a collection of assorted marine bacterial isolates. We also used the denaturing gradient gel electrophoresis (DGGE) approach in environmental samples to evaluate their ability to yield clear and diverse band patterns corresponding to Bacteroidetes phylotypes. Among the primers tested, CF968R did not provide satisfactory results in DGGE, although it exhibited the highest in silico coverage for Flavobacteria. Primers CFB560 and CFB555 presented undesirable features, such as requiring nested protocols or presence of degeneracies. Finally, the new primer CF418 and primer CF319a were used to explore the Bacteroidetes dynamics throughout a 1-year cycle in Mediterranean coastal waters (Blanes Bay Microbial Observatory). Both primers provided clear and diverse banding patterns, but the low specificity of CF319a was evidenced by 83.3 % of the bands sequenced corresponding to nontarget taxa. The satisfactory DGGE banding patterns and the wide diversity of sequences retrieved from DGGE bands with primer CF418 prove it to be a valuable alternative for the study of Bacteroidetes communities, recovering a wide range of phylotypes within the group.

The ecological significance of the marine bacterial populations distinguishable by flow cytometry on the basis of the fluorescence (FL) of their nucleic acid (NA) content and proxies of cell size (such as side scatter, SSC) remains largely unknown. Some studies have suggested that cells with high NA (HNA) content and high SSC (HS) represent the active members of the community, while the low NA (LNA) cells are inactive members of the same phylogenetic groups. But group-specific activity measurements and phylogenetic assignment after cell sorting have suggested this is not be the case, particularly in open-ocean communities. To test the extent to which the different NA subgroups are similar, and consequently the extent to which they likely have similar ecological and biogeochemical roles in the environment, we analysed the phylogenetic composition of three populations after cell sorting [high NA-high SC (HNA-HS), high NA-low SC (HNA-LS), low NA (LNA)] by 454 pyrosequencing in two contrasting periods of the year in NW Mediterranean coastal waters (BBMO, Blanes Bay Microbial Observatory) where these three populations have recurrent seasonal patterns. Statistical analyses showed that summer and winter samples were significantly different and, importantly, the sorted populations within a sample were composed of different taxa. The majority of taxa were associated with one NA fraction only, and the degree of overlap (i.e. OTUs present simultaneously in 2 fractions) between HNA and LNA and between summer and winter communities was very small. Rhodobacterales, SAR116 and Bacteroidetes contributed primarily to the HNA fraction, whereas other groups such as SAR11 and SAR86 contributed largely to the LNA fractions. Gammaproteobacteria other than SAR86 showed less preference for one particular NA fraction. An increase in diversity was observed from the LNA to the HNA-HS fraction for both sample dates. Our results suggest that, in Blanes Bay, flow cytometric signatures of natural communities track their phylogenetic composition., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

We analyzed the regional distribution of bulk heterotrophic prokaryotic activity (leucine incorporation) and selected single-cell parameters (cell viability and nucleic acid content) as parameters for microbial functioning, as well as bacterial and archaeal community structure in the epipelagic (0 to 200 m) and mesopelagic (200 to 1,000 m) subtropical Northeast Atlantic Ocean. We selectively sampled three contrasting regions covering a wide range of surface productivity and oceanographic properties within the same basin: (i) the eddy field south of the Canary Islands, (ii) the open-ocean NE Atlantic Subtropical Gyre, and (iii) the upwelling filament off Cape Blanc. In the epipelagic waters, a high regional variation in hydrographic parameters and bacterial community structure was detected, accompanied, however, by a low variability in microbial functioning. In contrast, mesopelagic microbial functioning was highly variable between the studied regions despite the homogeneous abiotic conditions found therein. More microbial functioning parameters indicated differences among the three regions within the mesopelagic (i.e., viability of cells, nucleic acid content, cell-specific heterotrophic activity, nanoflagellate abundance, prokaryote-to-nanoflagellate abundance ratio) than within the epipelagic (i.e., bulk activity, nucleic acid content, and nanoflagellate abundance) waters. Our results show that the mesopelagic realm in the Northeast Atlantic is, in terms of microbial activity, more heterogeneous than its epipelagic counterpart, probably linked to mesoscale hydrographical variations.

The sensitivity of coastal marine bacterioplankton to natural photosynthetically active radiation (PAR, 400-700 nm) and ultraviolet radiation (UVR, 280-400 nm) was evaluated in five experiments over a seasonal cycle in the Blanes Bay, NW Mediterranean Sea. Exposure to natural solar radiation generally inhibited bulk bacterial activities or damaged membrane integrity when irradiances were high (i.e. spring and summer experiments) and, in general, UVB (280-320 nm) accounted for most of the inhibition. When assessing activity ((3) H-leucine uptake) at the single-cell level by microautoradiography and rRNA gene probing, seasonally varying responses and sensitivities were found among bacterial groups. While autumn and winter irradiances seemed too low to cause changes in activity, variable effects were found in spring and summer. SAR11 was consistently inhibited by UVR and PAR exposure, whereas Gammaproteobacteria and Bacteroidetes showed higher resistance. Roseobacter, Synechococcus and the NOR5 clade were occasionally photostimulated in their activity, mainly because of PAR. Our results indicate that a component of seasonality exists in the bacterial responses to solar radiation, which vary not only depending on the irradiance and the spectral characteristics, but also on the previous light history and the taxonomic composition of the community., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Growth is one of the basic attributes of any living organism. Surprisingly, the growth rates of marine bacterioplankton are only poorly known. Current data suggest that marine bacteria grow relatively slowly, having generation times of several days. However, some bacterial groups, such as the aerobic anoxygenic phototrophic (AAP) bacteria, have been shown to grow much faster. Two manipulation experiments, in which grazing, viruses, and resource competition were reduced, were conducted in the coastal Mediterranean Sea (Blanes Bay Microbial Observatory). The growth rates of AAP bacteria and of several important phylogenetic groups (the Bacteroidetes, the alphaproteobacterial groups Roseobacter and SAR11, and the Gammaproteobacteria group and its subgroups the Alteromonadaceae and the NOR5/OM60 clade) were calculated from changes in cell numbers in the manipulation treatments. In addition, we examined the role that top-down (mortality due to grazers and viruses) and bottom-up (resource availability) factors play in determining the growth rates of these groups. Manipulations resulted in an increase of the growth rates of all groups studied, but its extent differed largely among the individual treatments and among the different groups. Interestingly, higher growth rates were found for the AAP bacteria (up to 3.71 day⁻¹) and for the Alteromonadaceae (up to 5.44 day⁻¹), in spite of the fact that these bacterial groups represented only a very low percentage of the total prokaryotic community. In contrast, the SAR11 clade, which was the most abundant group, was the slower grower in all treatments. Our results show that, in general, the least abundant groups exhibited the highest rates, whereas the most abundant groups were those growing more slowly, indicating that some minor groups, such the AAP bacteria, very likely contribute much more to the recycling of organic matter in the ocean than what their abundances alone would predict.

An understanding of the distribution of colored dissolved organic matter (CDOM) in the oceans and its role in the global carbon cycle requires a better knowledge of the colored materials produced and consumed by marine phytoplankton and bacteria. In this work, we examined the net uptake and release of CDOM by a natural bacterial community growing on DOM derived from four phytoplankton species cultured under axenic conditions. Fluorescent humic-like substances exuded by phytoplankton (excitation/emission [Ex/Em] wavelength, 310 nm/392 nm; Coble's peak M) were utilized by bacteria in different proportions depending on the phytoplankton species of origin. Furthermore, bacteria produced humic-like substances that fluoresce at an Ex/Em wavelength of 340 nm/440 nm (Coble's peak C). Differences were also observed in the Ex/Em wavelengths of the protein-like materials (Coble's peak T) produced by phytoplankton and bacteria. The induced fluorescent emission of CDOM produced by prokaryotes was an order of magnitude higher than that of CDOM produced by eukaryotes. We have also examined the final compositions of the bacterial communities growing on the exudates, which differed markedly depending on the phytoplankton species of origin. Alteromonas and Roseobacter were dominant during all the incubations on Chaetoceros sp. and Prorocentrum minimum exudates, respectively. Alteromonas was the dominant group growing on Skeletonema costatum exudates during the exponential growth phase, but it was replaced by Roseobacter afterwards. On Micromonas pusilla exudates, Roseobacter was replaced by Bacteroidetes after the exponential growth phase. Our work shows that fluorescence excitation-emission matrices of CDOM can be a helpful tool for the identification of microbial sources of DOM in the marine environment, but further studies are necessary to explore the association of particular bacterial groups with specific fluorophores.

The distribution of aerobic anoxygenic phototrophs (AAPs) was surveyed in various regions of the Mediterranean Sea in spring and summer. These phototrophic bacteria were present within the euphotic layer at all sampled stations. The AAP abundances increased with increasing trophic status ranging from 2.5 × 10(3) cells per ml in oligotrophic Eastern Mediterranean up to 90 × 10(3) cells per ml in the Bay of Villefranche. Aerobic anoxygenic phototrophs made up on average 1-4% of total prokaryotes in low nutrient areas, whereas in coastal and more productive stations these organisms represented 3-11% of total prokaryotes. Diel bacteriochlorophyll a decay measurements showed that AAP community in the Western Mediterranean grew rapidly, at rates from 1.13 to 1.42 day(-1). The lower AAP abundances registered in the most oligotrophic waters suggest that they are relatively poor competitors under nutrient limiting conditions. Instead, AAPs appear to be metabolically active organisms, which thrive better in more eutrophic environments providing the necessary substrates to maintain high growth rates., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

The suitability of applying empirical conversion factors (eCFs) to determine bacterial biomass production remains unclear because seawater cultures are usually overtaken by phylotypes that are not abundant in situ. While eCFs vary across environments, it has not been tested whether differences in eCFs are driven by changes in bacterial community composition or by in situ environmental conditions. We carried out seawater cultures throughout a year to analyse the correlation between eCFs and bacterial community structure, analysed by catalysed reporter deposition fluorescence in situ hybridization. Gammaproteobacteria usually dominated seawater cultures, but their abundance exhibited a wide range (25-73% of cell counts) and significantly increased with inorganic nutrient enrichment. Flavobacteria were less abundant but increased up to 40% of cells counts in winter seawater cultures, when in situ chlorophyll a was high. The correlations between eCFs and the abundance of the main broad phylogenetic groups (Gamma-, Alphaproteobacteria and Flavobacteria) were significant, albeit weak, while more specific groups (Alteromonadaceae and Rhodobacteraceae) were not significantly correlated. Our results show that the frequent development of the fast-growing group Alteromonadaceae in seawater cultures does not strongly drive the observed variations in eCFs. Rather, the results imply that environmental conditions and the growth of specific phylotypes interact to determine eCFs., (© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.)

To investigate the effects of mesoscale eddies on prokaryotic assemblage structure and activity, we sampled two cyclonic eddies (CEs) and two anticyclonic eddies (AEs) in the permanent eddy-field downstream the Canary Islands. The eddy stations were compared with two far-field (FF) stations located also in the Canary Current, but outside the influence of the eddy field. The distribution of prokaryotic abundance (PA), bulk prokaryotic heterotrophic activity (PHA), various indicators of single-cell activity (such as nucleic acid content, proportion of live cells, and fraction of cells actively incorporating leucine), as well as bacterial and archaeal community structure were determined from the surface to 2000 m depth. In the upper epipelagic layer (0-200 m), the effect of eddies on the prokaryotic community was more apparent, as indicated by the higher PA, PHA, fraction of living cells, and percentage of active cells incorporating leucine within eddies than at FF stations. Prokaryotic community composition differed also between eddy and FF stations in the epipelagic layer. In the mesopelagic layer (200-1000 m), there were also significant differences in PA and PHA between eddy and FF stations, although in general, there were no clear differences in community composition or single-cell activity. The effects on prokaryotic activity and community structure were stronger in AE than CE, decreasing with depth in both types of eddies. Overall, both types of eddies show distinct community compositions (as compared with FF in the epipelagic), and represent oceanic 'hotspots' of prokaryotic activity (in the epi- and mesopelagic realms).

Previsions of a warmer ocean as a consequence of climatic change point to a 2-6 degrees C temperature rise during this century in surface oceanic waters. Heterotrophic bacteria occupy the central position of the marine microbial food web, and their metabolic activity and interactions with other compartments within the web are regulated by temperature. In particular, key ecosystem processes like bacterial production (BP), respiration (BR), growth efficiency and bacterial-grazer trophic interactions are likely to change in a warmer ocean. Different approaches can be used to predict these changes. Here we combine evidence of the effects of temperature on these processes and interactions coming from laboratory experiments, space-for-time substitutions, long-term data from microbial observatories and theoretical predictions. Some of the evidence we gathered shows opposite trends to warming depending on the spatio-temporal scale of observation, and the complexity of the system under study. In particular, we show that warming (i) increases BR, (ii) increases bacterial losses to their grazers, and thus bacterial-grazer biomass flux within the microbial food web, (iii) increases BP if enough resources are available (as labile organic matter derived from phytoplankton excretion or lysis), and (iv) increases bacterial losses to grazing at lower rates than BP, and hence decreasing the proportion of production removed by grazers. As a consequence, bacterial abundance would also increase and reinforce the already dominant role of microbes in the carbon cycle of a warmer ocean.

Catalysed reported deposition-FISH and clone libraries indicated that Roseobacter, followed by Bacteroidetes, and some gammaproteobacterial groups such as SAR86, dominated the composition of bacterioplankton in Ría de Vigo, NW Spain, in detriment to SAR11 (almost absent in this upwelling ecosystem). Since we sampled four times during the year, we observed pronounced changes in the structure of each bacterioplankton component, particularly for the Roseobacter lineage. We suggest that such variations in the coastal upwelling ecosystem of Ría de Vigo were associated with the characteristic phytoplankton communities of the four different hydrographical situations: winter mixing, spring bloom, summer stratification, and autumn upwelling. We retrieved new sequences among the major marine bacterial lineages, particularly among Roseobacter, SAR11, and especially SAR86. The spring community was dominated by two Roseobacter clades that had previously been related to phytoplankton blooms. In the other seasons, communities with higher diversity than the spring one were detected.

We demonstrate here results showing that bottom-up and top-down control mechanisms can operate simultaneously and in concert in marine microbial food webs, controlling prokaryote diversity by a combination of viral lysis and substrate limitation. Models in microbial ecology predict that a shift in the type of bacterial growth rate limitation is expected to have a major effect on species composition within the community of bacterial hosts, with a subsequent shift in the composition of the viral community. Only moderate effects would, however, be expected in the absolute number of coexisting virus-host pairs. We investigated these relationships in nutrient-manipulated systems, under simulated in situ conditions. There was a strong correlation in the clustering of the viral and bacterial community data supporting the existence of an important link between the bacterial and viral communities. As predicted, the total number of viral populations was the same in all treatments, while the composition of the viral community varied. Our results support the theoretical prediction that there is one control mechanism for the number of niches for coexisting virus-host pairs (top-down control), and another mechanism that controls which virus-host pairs occupy these niches (bottom-up control).

Prokaryotic extracellular enzymatic activity, abundance, heterotrophic production and respiration were determined in the meso- and bathypelagic (sub)tropical North Atlantic. While prokaryotic heterotrophic production (PHP) decreased from the lower euphotic layer to the bathypelagic waters by two orders of magnitude, prokaryotic abundance and cell-specific PHP decreased only by one order of magnitude. In contrast to cell-specific PHP, cell-specific extracellular enzymatic activity (alpha- and beta-glucosidase, leucine aminopeptidase, alkaline phosphatase) increased with depth as did cell-specific respiration rates. Cell-specific alkaline phosphatase activity increased from the intermediate water masses to the deep waters up to fivefold. Phosphate concentrations, however, varied only by a factor of two between the different water masses, indicating that phosphatase activity is not related to phosphate availability in the deep waters. Generally, cell-specific extracellular enzymatic activities were inversely related to cell-specific prokaryotic leucine incorporation. Thus, it is apparent that the utilization of deep ocean organic matter is linked to higher cell-specific extracellular enzymatic activity and respiration and lower cell-specific PHP than in surface waters.

We used mesocosm experiments to study the bacterioplankton community in a highly dynamic coastal ecosystem during four contrasting periods of the seasonal cycle: winter mixing, spring phytoplankton bloom, summer stratification and autumn upwelling. A correlation approach was used in order to measure the degree of coupling between the dynamics of major bacterial groups, heterotrophic carbon cycling and environmental factors. We used catalysed reporter deposition-fluorescence in situ hybridization to follow changes in the relative abundance of the most abundant groups of bacteria (Alphaproteobacteria, Gammaproteobacteria and Bacteroidetes). Bacterial carbon flux-related variables included bacterial standing stock, bacterial production and microbial respiration. The environmental factors included both, biotic variables such as chlorophyll-a concentration, primary production, phytoplankton extracellular release, and abiotic variables such as the concentration of dissolved inorganic and organic nutrients. Rapid shifts in the dominant bacterial groups occurred associated to environmental changes and bacterial bulk functions. An alternation between Alphaproteobacteria and Bacteroidetes was observed associated to different phytoplankton growth phases. The dominance of the group Bacteroidetes was related to high bacterial biomass and production. We found a significant, non-spurious, linkage between the relative abundances of major bacterial groups and bacterial carbon cycling. Our results suggest that bacteria belonging to these major groups could actually share a function in planktonic ecosystems.

An annual seasonal cycle of composition of a bacterioplankton community in an oligotrophic coastal system was studied by denaturing gradient gel electrophoresis (DGGE) using five different primer sets. Analysis of DGGE fingerprints showed that primer set 357fGC-907rM grouped samples according to seasons. Additionally, we used the set of 16S rRNA genes archived in the RDPII database to check the percentage of perfect matches of each primer for the most abundant bacterial groups inhabiting coastal plankton communities. Overall, primer set 357fGC-907rM was the most suitable for the routine use of PCR-DGGE analyses in this environment.

We analyzed the contributions of different heterotrophic bacterial groups to the uptake of several low-molecular weight compounds during a seasonal cycle on the northwestern Mediterranean coast (Blanes Bay Microbial Observatory). The bacterial assemblage structure had been shown to change substantially year-round for this site, but whether changes in the activities of the different bacterial groups also occurred on the seasonal scale was unknown. Microautoradiography combined with catalyzed reporter deposition fluorescence in situ hybridization was used to analyze the patterns of glucose, amino acid, and ATP uptake by different bacterial groups. Gammaproteobacteria and Bacteroidetes were not very active in the uptake of glucose at any time of the year (<10% of cells were active) compared to Alphaproteobacteria (generally >20% of cells were active). Dissolved free amino acids were taken up considerably by Alphaproteobacteria and Gammaproteobacteria but not by Bacteroidetes. Relatively high percentages of cells of the three broad phylogenetic groups actively took up ATP, which could be related to the important phosphorous limitation of bacterial production during most of the year in Blanes Bay. The contribution of SAR11 to the uptake of the monomers was variable year-round, generally with fewer than 30% of the cells being active. By contrast, Roseobacter were highly overrepresented in the uptake of all the substrates throughout all the year, with more than 50% of cells being active in all the samples and for all substrates. Our results suggest that substantial changes in the activity of some phylogenetic groups of bacteria occur throughout the year.

We combined denaturing gradient gel electrophoresis (DGGE), catalysed reporter deposition-FISH (CARD-FISH) and clone libraries to investigate the seasonality of the bacterial assemblage composition in north-west Mediterranean coastal waters. DGGE analysis indicated that bacterial diversity changed gradually throughout the year, although with a clear distinction of the summer period. Alphaproteobacteria were the dominant group on an annual basis [29% of the DAPI (4',6-diamidino-2-phenylindole) counts by CARD-FISH, and 70% of the bacterial clones]. The SAR11 clade was most abundant during spring and summer (>20% of DAPI counts), while the Roseobacter clade was abundant primarily in winter and spring (up to 7% of DAPI counts). The phylum Bacteroidetes constituted the second most important group and was quantitatively uniform throughout the year (average 11% of the DAPI counts). Gammaproteobacteria showed a peak during summer (8% of DAPI counts), when most of them belonged to the NOR5 cluster. Clone libraries and CARD-FISH showed reasonable agreement in the quantitative proportions of Bacteroidetes and Gammaproteobacteria, but Alphaproteobacteria were overrepresented in clone libraries. Sequencing of the most predominant DGGE bands failed to detect the SAR11 group despite their high abundance. The combination of the three molecular approaches allowed a comprehensive assessment of seasonal changes in bacterial diversity.

Dimethylsulfoniopropionate (DMSP) accounts for most of the organic sulfur fluxes from primary to secondary producers in marine microbial food webs. Incubations of natural communities and axenic cultures with radio-labeled DMSP showed that dominant phytoplankton groups of the ocean, the unicellular cyanobacteria Prochlorococcus and Synechococcus and diatoms, as well as heterotrophic bacteria take up and assimilate DMSP sulfur, thus diverting a proportion of plankton-produced organic sulfur from emission into the atmosphere.

We studied the effects of natural sunlight on heterotrophic marine bacterioplankton in short-term experiments. We used a single-cell level approach involving flow cytometry combined with physiological probes and microautoradiography to determine sunlight effects on the activity and integrity of the cells. After 4 h of sunlight exposure, most bacterial cells maintained membrane integrity and viability as assessed by the simultaneous staining with propidium iodide and SYBR green I. In contrast, a significant inhibition of heterotrophic bacterial activity was detected, measured by 5-cyano-2,3 ditolyl tetrazolium chloride reduction and leucine incorporation. We applied microautoradiography combined with catalyzed reporter deposition-fluorescence in situ hybridization to test the sensitivity of the different bacterial groups naturally occurring in the Northwestern Mediterranean to sunlight. Members of the Gammaproteobacteria and Bacteroidetes groups appeared to be highly resistant to solar radiation, with small changes in activity after exposure. On the contrary, Alphaproteobacteria bacteria were more sensitive to radiation as measured by the cell-specific incorporation of labeled amino acids, leucine, and ATP. Within Alphaproteobacteria, bacteria belonging to the Roseobacter group showed higher resistance than members of the SAR11 cluster. The activity of Roseobacter was stimulated by exposure to photosynthetic available radiation compared to the dark treatment. Our results suggest that UV radiation can significantly affect the in situ single-cell activity of bacterioplankton and that naturally dominating phylogenetic bacterial groups have different sensitivity to natural levels of incident solar radiation.

We tested the sensitivity of coastal picophytoplankton exposed to natural sunlight in short-term experiments. Cell abundance and cell-specific chlorophyll fluorescence were significantly reduced in Prochlorococcus spp. but not in Synechococcus, whereas picoeukaryotes had an intermediate response. These results are the first direct evidence of a differential sensitivity to sunlight of these ubiquitous marine members of unicellular phytoplankton.