Marta Sebastián

Determinants of community structure in the global plankton interactome

Species interaction networks are shaped by abiotic and biotic factors. Here, as part of the Tara Oceans project, we studied the photic zone interactome using environmental factors and organismal abundance profiles and found that environmental factors are incomplete predictors of community structure. We found associations across plankton functional types and phylogenetic groups to be nonrandomly distributed on the network and driven by both local and global patterns. We identified interactions among grazers, primary producers, viruses, and (mainly parasitic) symbionts and validated network-generated hypotheses using microscopy to confirm symbiotic relationships. We have thus provided a resource to support further research on ocean food webs and integrating biological components into ocean models.

The alkaline phosphatase PhoX is more widely distributed in marine bacteria than the classical PhoA

Phosphorus (P) is a vital nutrient for all living organisms and may control the growth of bacteria in the ocean. Bacteria induce alkaline phosphatases when inorganic phosphate (Pi) is insufficient to meet their P-requirements, and therefore bulk alkaline phosphatase activity measurements have been used to assess the P-status of microbial assemblages. In this study, the molecular basis of marine bacterial phosphatases and their potential role in the environment were investigated. We found that only a limited number of homologs to the classical Escherichia coli alkaline phosphatase (PhoA) were present in marine isolates in the Bacteroidetes and γ-proteobacteria lineages. In contrast, PhoX, a recently described phosphatase, was widely distributed among diverse bacterial taxa, including Cyanobacteria, and frequently found in the marine metagenomic Global Ocean Survey database. These taxa included ecologically important groups such as Roseobacter and Trichodesmium. PhoX was induced solely upon P-starvation and accounted for approximately 90% of the phosphatase activity in the model marine bacterium Silicibacter pomeroyi. Analysis of the available transcriptomic datasets and their corresponding metagenomes indicated that PhoX is more abundant than PhoA in oligotrophic marine environments such as the North Pacific Subtropical Gyre. Those analyses also revealed that PhoA may be important when Bacteroidetes are abundant, such as in algal bloom episodes. However, PhoX appears to be much more widespread. Its identification as a gene that mediates organic P acquisition in ecologically important groups, and as a marker of Pi-stress, constitutes an important step toward a better understanding of the marine P cycle.

Comparison of Growth Rates of Aerobic Anoxygenic Phototrophic Bacteria and Other Bacterioplankton Groups in Coastal Mediterranean Waters

ABSTRACT 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.

Lipid remodelling is a widespread strategy in marine heterotrophic bacteria upon phosphorus deficiency.

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.

Bacterioplankton groups involved in the uptake of phosphate and dissolved organic phosphorus in a mesocosm experiment with P-starved Mediterranean waters

The use of inorganic phosphate (Pi) and dissolved organic phosphorus (DOP) by different bacterial groups was studied in experimental mesocosms of P-starved eastern Mediterranean waters in the absence (control mesocosms) and presence of additional Pi (P-amended mesocosms). The low Pi turnover times in the control mesocosms and the increase in heterotrophic prokaryotic abundance and production upon Pi addition confirmed that the bacterial community was originally P-limited. The bacterioplankton groups taking up Pi and DOP were identified by means of microautoradiography combined with catalysed reporter deposition fluorescence in situ hybridization. Incubations with leucine were also performed for comparative purposes. All the probe-identified groups showed a high percentage of cells taking up Pi and DOP in the control, P-limited, mesocosms throughout the experiment. However, in response to Pi addition two contrasting scenarios in Pi use were observed: (i) on day 1 of the experiment Pi addition caused a clear reduction in the percentage of SAR11 cells taking up Pi, whereas Gammaproteobacteria, Roseobacter and Bacteroidetes showed similar percentages to the ones in the control mesocosms and (ii) on day 4 of the experiment, probably when the bacterial community had fully responded to the P input, all the probe-identified groups showed low percentages of cells taking up the substrate as compared with the control mesocosms. These differences are likely related to different P requirements among the bacterial groups and point out to the existence of two contrasting strategies in P use.

Heterogeneity in the nutrient limitation of different bacterioplankton groups in the Eastern Mediterranean Sea

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.

Role of the phosphatase PhoX in the phosphorus metabolism of the marine bacterium Ruegeria pomeroyi DSS-3

Marine microbes are adapted to surviving in a variable phosphorus (P) environment. This adaptation frequently involves the presence of periplasmic or cell membrane-associated enzymes that enable them access to alternative sources of P when phosphate is depleted. In a recent study we identified the phosphatase PhoX as an enzyme that may be essential in mediating organic P acquisition in the ocean. Here we have investigated the role of this enzyme in the utilization of different P sources, using as a model the marine bacterium Ruegeria pomeroyi DSS-3. Although our previous study had demonstrated that PhoX accounts for more than 90% of the alkaline phosphatase (APase) activity in R. pomeroyi, a PhoX mutant strain was able to grow on monophosphate esters at the same rate as the wild type. Nevertheless, further APase kinetic analyses with both strains demonstrated that the Km of the wild-type strain was an order of magnitude lower than the mutant strain, indicating that PhoX is crucial for the use of these substrates at low concentrations, typically found in seawater. We also showed that PhoX is required for efficient hydrolysation of nucleotides like ADP and ATP.

Enzymatic assay of marine bacterial phosphatases by capillary electrophoresis with laser-induced fluorescence detection†

Microbial ectoenzyme activities in aquatic environments are important determinants of polymer hydrolysis and indicators of the state of microbial carbon, nitrogen, and phosphorus nutrition. Marine ectoenzymes are found on the cell surface or in the periplasmic space of gram‐negative heterotrophic bacteria. Phosphatases, which remove phosphate groups from substrates, are one example of an ectoenzyme. Enzyme assays based on‐capillary electrophoresis (CE) take advantage of CEs high‐efficiency separation, extremely low sample volume requirements, and its ability to electrophoretically mix and separate zones of enzymes, substrates, and products all in one experimental run. CE has better resolving power and, when utilized with laser‐induced fluorescence (LIF) detection, it is more sensitive than chromatography. CE‐LIF is a promising tool for determining different phosphatases within a single microbial strain as well as the functional diversity between strains. In this study, four bacterial strains were studied (Shewanella sp., TW7, BB2AT2, and Vibrio alginolyticus) with each yielding at least one phosphatase that was kinetically characterized. Km values were calculated and found to be in the range of 0.0725–3.35 µM, whereas Vmax values ranged from 1.02×10−3 to 1.05×10−2 µM/min. The large range of values demonstrates differences among the phosphatases, suggesting different roles for each phosphatase not only between the species but also within a single bacterial species. This can have the important implications for organic matter processing in the sea.

Prokaryotic functional gene diversity in the sunlit ocean: Stumbling in the dark

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.

Different processes shape prokaryotic and picoeukaryotic assemblages in the sunlit ocean microbiome

The smallest members of the sunlit-ocean microbiome (prokaryotes and picoeukaryotes) participate in a plethora of ecosystem functions with planetary-scale effects. Understanding the processes determining the spatial turnover of this assemblage can help us better comprehend the links between microbiome species composition and ecosystem function. Ecological theory predicts that selection, dispersal and drift are main drivers of species distributions, yet, the relative quantitative importance of these ecological processes in structuring the surface-ocean microbiome is barely known. Here we quantified the role of selection, dispersal and drift in structuring surface-ocean prokaryotic and picoeukaryotic assemblages by using community DNA-sequence data collected during the global Malaspina expedition. We found that dispersal limitation was the dominant process structuring picoeukaryotic communities, while a balanced combination of dispersal limitation, selection and drift shaped prokaryotic counterparts. Subsequently, we determined the agents exerting abiotic selection as well as the spatial patterns emerging from the action of different ecological processes. We found that selection exerted via temperature had a strong influence on the structure of prokaryotic communities, particularly on species co-occurrences, a pattern not observed among communities of picoeukaryotes. Other measured abiotic variables had limited selective effects on microbiome structure. Picoeukaryotes presented a higher differentiation between neighbouring communities and a higher distance-decay when compared to prokaryotes, agreeing with their higher dispersal limitation. Finally, drift seemed to have a limited role in structuring the sunlit-ocean microbiome. The different predominance of ecological processes acting on particular subsets of the ocean microbiome suggests uneven responses to environmental change. SIGNIFICANCE STATEMENT The global ocean contains one of the largest microbiomes on Earth and changes on its structure can impact the functioning of the biosphere. Yet, we are far from understanding the mechanisms that structure the global ocean microbiome, that is, the relative importance of environmental selection, dispersal and random events (drift). We evaluated the role of these processes at the global scale, based on data derived from a circumglobal expedition and found that these ecological processes act differently on prokaryotes and picoeukaryotes, two of the main components of the ocean microbiome. Our work represents a significant contribution to understand the assembly of marine microbial communities, providing also insights on the links between ecological mechanisms, microbiome structure and ecosystem function.