Single-cell analysis in hypersaline brines predicts a water-activity limit of microbial anabolic activity.

Hypersaline brines provide excellent opportunities to study extreme microbial life. Here, we investigated anabolic activity in nearly 6000 individual cells from solar saltern sites with water activities (aw) ranging from 0.982 to 0.409 (seawater to extreme brine). Average anabolic activity decreased exponentially with aw, with nuanced trends evident at the single-cell level: The proportion of active cells remained high (>50%) even after NaCl saturation, and subsets of cells spiked in activity as aw decreased. Intracommunity heterogeneity in activity increased as seawater transitioned to brine, suggesting increased phenotypic heterogeneity with increased physiological stress. No microbial activity was detected in the 0.409-aw brine (an MgCl2-dominated site) despite the presence of cell-like structures. Extrapolating our data, we predict an aw limit for detectable anabolic activity of 0.540, which is beyond the currently accepted limit of life based on cell division. This work demonstrates the utility of single-cell, metabolism-based techniques for detecting active life and expands the potential habitable space on Earth and beyond.

Temperature enhances the functional diversity of dissolved organic matter utilization by coastal marine bacteria.

Although bulk bacterial metabolism in response to temperature has been determined for different oceanic regions, the impact of temperature on the functional diversity of dissolved organic matter (DOM) utilization has been largely unexplored. Here, we hypothesized that besides modifying the rates of carbon utilization, temperature can also alter the diversity of substrates utilized. The patterns of utilization of 31 model DOM compounds (as represented in Biolog EcoPlate™) by bacterioplankton were assessed using inocula from surface waters of the southern Bay of Biscay continental shelf over 1 year. Bacteria utilized more polymers and carbohydrates in late spring and summer than in winter, likely reflecting changes in substrate availability linked to the release and accumulation of DOM in phytoplankton post-bloom conditions. Seawater temperature correlated positively with the number of substrates utilized (i.e. functional richness) and this relationship was maintained in monthly experimental incubations spanning 3°C below and above in situ values. The enhancement of functional richness with experimental warming displayed a unimodal response to ambient temperature, peaking at 16°C. This temperature acted as a threshold separating nutrient-sufficient from nutrient-deficient conditions at the study site, suggesting that trophic conditions will be critical in the response of microbial DOM utilization to future warming.

Efficient carbon and nitrogen transfer from marine diatom aggregates to colonizing bacterial groups.

Bacterial degradation of sinking diatom aggregates is key for the availability of organic matter in the deep-ocean. Yet, little is known about the impact of aggregate colonization by different bacterial taxa on organic carbon and nutrient cycling within aggregates. Here, we tracked the carbon (C) and nitrogen (N) transfer from the diatom Leptocylindrus danicus to different environmental bacterial groups using a combination of 13C and 15N isotope incubation (incubated for 72 h), CARD-FISH and nanoSIMS single-cell analysis. Pseudoalteromonas bacterial group was the first colonizing diatom-aggregates, succeeded by the Alteromonas group. Within aggregates, diatom-attached bacteria were considerably more enriched in 13C and 15N than non-attached bacteria. Isotopic mass balance budget indicates that both groups showed comparable levels of diatom C in their biomass, accounting for 19 ± 7% and 15 ± 11%, respectively. In contrast to C, bacteria of the Alteromonas groups showed significantly higher levels of N derived from diatoms (77 ± 28%) than Pseudoalteromonas (47 ± 17%), suggesting a competitive advantage for Alteromonas in the N-limiting environments of the deep-sea. Our results imply that bacterial succession within diatom aggregates may largely impact taxa-specific C and N uptake, which may have important consequences for the quantity and quality of organic matter exported to the deep ocean.

Characterizing the "fungal shunt": Parasitic fungi on diatoms affect carbon flow and bacterial communities in aquatic microbial food webs.

Microbial interactions in aquatic environments profoundly affect global biogeochemical cycles, but the role of microparasites has been largely overlooked. Using a model pathosystem, we studied hitherto cryptic interactions between microparasitic fungi (chytrid Rhizophydiales), their diatom host Asterionella, and cell-associated and free-living bacteria. We analyzed the effect of fungal infections on microbial abundances, bacterial taxonomy, cell-to-cell carbon transfer, and cell-specific nitrate-based growth using microscopy (e.g., fluorescence in situ hybridization), 16S rRNA gene amplicon sequencing, and secondary ion mass spectrometry. Bacterial abundances were 2 to 4 times higher on individual fungal-infected diatoms compared to healthy diatoms, particularly involving Burkholderiales. Furthermore, taxonomic compositions of both diatom-associated and free-living bacteria were significantly different between noninfected and fungal-infected cocultures. The fungal microparasite, including diatom-associated sporangia and free-swimming zoospores, derived ∼100% of their carbon content from the diatom. By comparison, transfer efficiencies of photosynthetic carbon were lower to diatom-associated bacteria (67 to 98%), with a high cell-to-cell variability, and even lower to free-living bacteria (32%). Likewise, nitrate-based growth for the diatom and fungi was synchronized and faster than for diatom-associated and free-living bacteria. In a natural lacustrine system, where infection prevalence reached 54%, we calculated that 20% of the total diatom-derived photosynthetic carbon was shunted to the parasitic fungi, which can be grazed by zooplankton, thereby accelerating carbon transfer to higher trophic levels and bypassing the microbial loop. The herein termed "fungal shunt" can thus significantly modify the fate of photosynthetic carbon and the nature of phytoplankton-bacteria interactions, with implications for diverse pelagic food webs and global biogeochemical cycles.

Seasonal dynamics of natural Ostreococcus viral infection at the single cell level using VirusFISH.

Ostreococcus is a cosmopolitan marine genus of phytoplankton found in mesotrophic and oligotrophic waters, and the smallest free-living eukaryotes known to date, with a cell diameter close to 1 µm. Ostreococcus has been extensively studied as a model system to investigate viral-host dynamics in culture, yet the impact of viruses in naturally occurring populations is largely unknown. Here, we used Virus Fluorescence in situ Hybridization (VirusFISH) to visualize and quantify viral-host dynamics in natural populations of Ostreococcus during a seasonal cycle in the central Cantabrian Sea (Southern Bay of Biscay). Ostreococcus were predominantly found during summer and autumn at surface and 50 m depth, in coastal, mid-shelf and shelf waters, representing up to 21% of the picoeukaryotic communities. Viral infection was only detected in surface waters, and its impact was variable but highest from May to July and November to December, when up to half of the population was infected. Metatranscriptomic data available from the mid-shelf station unveiled that the Ostreococcus population was dominated by the species O. lucimarinus. This work represents a proof of concept that the VirusFISH technique can be used to quantify the impact of viruses on targeted populations of key microbes from complex natural communities.

Microbial diversity and activity in Southern California salterns and bitterns: analogues for remnant ocean worlds.

Concurrent osmotic and chaotropic stress make MgCl2 -rich brines extremely inhospitable environments. Understanding the limits of life in these brines is essential to the search for extraterrestrial life on contemporary and relict ocean worlds, like Mars, which could host similar environments. We sequenced environmental 16S rRNA genes and quantified microbial activity across a broad range of salinity and chaotropicity at a Mars-analogue salt harvesting facility in Southern California, where seawater is evaporated in a series of ponds ranging from kosmotropic NaCl brines to highly chaotropic MgCl2 brines. Within NaCl brines, we observed a proliferation of specialized halophilic Euryarchaeota, which corresponded closely with the dominant taxa found in salterns around the world. These communities were characterized by very slow growth rates and high biomass accumulation. As salinity and chaotropicity increased, we found that the MgCl2 -rich brines eventually exceeded the limits of microbial activity. We found evidence that exogenous genetic material is preserved in these chaotropic brines, producing an unexpected increase in diversity in the presumably sterile MgCl2 -saturated brines. Because of their high potential for biomarker preservation, chaotropic brines could therefore serve as repositories of genetic biomarkers from nearby environments (both on Earth and beyond) making them prime targets for future life-detection missions.

Light supports cell-integrity and growth rates of taxonomically diverse coastal photoheterotrophs.

Despite the widespread distribution of proteorhodopsin (PR)-containing bacteria in the oceans, the use of light-derived energy to promote bacterial growth has only been shown in a few bacterial isolates, and there is a paucity of data describing the metabolic effects of light on environmental photoheterotrophic taxa. Here, we assessed the effects of light on the taxonomic composition, cell integrity and growth responses of microbial communities in monthly incubations between spring and autumn under different environmental conditions. The photoheterotrophs expressing PR in situ were dominated by Pelagibacterales and SAR116 in July and November, while members of Euryarchaeota, Gammaproteobacteria and Bacteroidetes dominated the PR expression in spring. Cell-membrane integrity decreased under dark conditions throughout most of the assessment, with maximal effects in summer, under low-nutrient conditions. A positive effect of light on growth was observed in one incubation (out of nine), coinciding with a declining phytoplankton bloom. Light-enhanced growth was found in Gammaproteobacteria (Alteromonadales) and Bacteroidetes (Polaribacter and Tenacibaculum). Unexpectedly, some Pelagibacterales also exhibited higher growth rates under light conditions. We propose that the energy harvested by PRs helps to maintain cell viability in dominant coastal photoheterotrophic oligotrophs while promoting the growth of some widespread taxa benefiting from the decline of phytoplankton blooms.

Warming the phycosphere: Differential effect of temperature on the use of diatom-derived carbon by two copiotrophic bacterial taxa.

Heterotrophic bacteria associated with microphytoplankton, particularly those colonizing the phycosphere, are major players in the remineralization of algal-derived carbon. Ocean warming might impact dissolved organic carbon (DOC) uptake by microphytoplankton-associated bacteria with unknown biogeochemical implications. Here, by incubating natural seawater samples at three different temperatures, we analysed the effect of experimental warming on the abundance and C and N uptake activity of Rhodobacteraceae and Flavobacteria, two bacterial groups typically associated with microphytoplankton. Using a nano-scale secondary ion mass spectrometry (nanoSIMS) single-cell analysis, we quantified the temperature sensitivity of these two taxonomic groups to the uptake of algal-derived DOC in the microphytoplankton associated fraction with 13 C-bicarbonate and 15 N-leucine as tracers. We found that cell-specific 13 C uptake was similar for both groups (~0.42 fg C h-1 µm-3 ), but Rhodobacteraceae were more active in 15 N-leucine uptake. Due to the higher abundance of Flavobacteria associated with microphytoplankton, this group incorporated fourfold more carbon than Rhodobacteraceae. Cell-specific 13 C uptake was influenced by temperature, but no significant differences were found for 15 N-leucine uptake. Our results show that the contribution of Flavobacteria and Rhodobacteraceae to C assimilation increased up to sixfold and twofold, respectively, with an increase of 3°C above ambient temperature, suggesting that warming may differently affect the contribution of distinct copiotrophic bacterial taxa to carbon cycling.

Temperature sensitivities of microbial plankton net growth rates are seasonally coherent and linked to nutrient availability.

Recent work suggests that temperature effects on marine heterotrophic bacteria are strongly seasonal, but few attempts have been made to concurrently assess them across trophic levels. Here, we estimated the temperature sensitivities (using activation energies, E) of autotrophic and heterotrophic microbial plankton net growth rates over an annual cycle in NE Atlantic coastal waters. Phytoplankton grew in winter and late autumn (0.41 ± 0.16 SE d-1 ) and decayed in the remaining months (-0.42 ± 0.10 d-1 ). Heterotrophic microbes shared a similar seasonality, with positive net growth for bacteria (0.14-1.48 d-1 ), while nanoflagellates had higher values (> 0.4 d-1 ) in winter and spring relative to the rest of the year (-0.46 to 0.29 d-1 ). Net growth rates activation energies showed similar dynamics in the three groups (-1.07 to 1.51 eV), characterized by maxima in winter, minima in summer and resumed increases in autumn. Microbial plankton E values were significantly correlated with nitrate concentrations as a proxy for nutrient availability. Nutrient-sufficiency (i.e., > 1 µmol l-1 nitrate) resulted in significantly higher activation energies of phytoplankton and heterotrophic nanoflagellates relative to nutrient-limited conditions. We suggest that only within spatio-temporal windows of both moderate bottom-up and top-down controls will temperature have a major enhancing effect on microbial growth.

Testing the metabolic theory of ecology with marine bacteria: different temperature sensitivity of major phylogenetic groups during the spring phytoplankton bloom.

Although temperature is a key driver of bacterioplankton metabolism, the effect of ocean warming on different bacterial phylogenetic groups remains unclear. Here, we conducted monthly short-term incubations with natural coastal bacterial communities over an annual cycle to test the effect of experimental temperature on the growth rates and carrying capacities of four phylogenetic groups: SAR11, Rhodobacteraceae, Gammaproteobacteria and Bacteroidetes. SAR11 was the most abundant group year-round as analysed by CARD-FISH, with maximum abundances in summer, while the other taxa peaked in spring. All groups, including SAR11, showed high temperature-sensitivity of growth rates and/or carrying capacities in spring, under phytoplankton bloom or post-bloom conditions. In that season, Rhodobacteraceae showed the strongest temperature response in growth rates, estimated here as activation energy (E, 1.43 eV), suggesting an advantage to outcompete other groups under warmer conditions. In summer E values were in general lower than 0.65 eV, the value predicted by the Metabolic Theory of Ecology (MTE). Contrary to MTE predictions, carrying capacity tended to increase with warming for all bacterial groups. Our analysis confirms that resource availability is key when addressing the temperature response of heterotrophic bacterioplankton. We further show that even under nutrient-sufficient conditions, warming differentially affected distinct bacterioplankton taxa.

Elevated temperature increases carbon and nitrogen fluxes between phytoplankton and heterotrophic bacteria through physical attachment.

Quantifying the contribution of marine microorganisms to carbon and nitrogen cycles and their response to predicted ocean warming is one of the main challenges of microbial oceanography. Here we present a single-cell NanoSIMS isotope analysis to quantify C and N uptake by free-living and attached phytoplankton and heterotrophic bacteria, and their response to short-term experimental warming of 4 °C. Elevated temperature increased total C fixation by over 50%, a small but significant fraction of which was transferred to heterotrophs within 12 h. Cell-to-cell attachment doubled the secondary C uptake by heterotrophic bacteria and increased secondary N incorporation by autotrophs by 68%. Warming also increased the abundance of phytoplankton with attached heterotrophs by 80%, and promoted C transfer from phytoplankton to bacteria by 17% and N transfer from bacteria to phytoplankton by 50%. Our results indicate that phytoplankton-bacteria attachment provides an ecological advantage for nutrient incorporation, suggesting a mutualistic relationship that appears to be enhanced by temperature increases.

Experimental Warming Decreases the Average Size and Nucleic Acid Content of Marine Bacterial Communities.

Organism size reduction with increasing temperature has been suggested as a universal response to global warming. Since genome size is usually correlated to cell size, reduction of genome size in unicells could be a parallel outcome of warming at ecological and evolutionary time scales. In this study, the short-term response of cell size and nucleic acid content of coastal marine prokaryotic communities to temperature was studied over a full annual cycle at a NE Atlantic temperate site. We used flow cytometry and experimental warming incubations, spanning a 6°C range, to analyze the hypothesized reduction with temperature in the size of the widespread flow cytometric bacterial groups of high and low nucleic acid content (HNA and LNA bacteria, respectively). Our results showed decreases in size in response to experimental warming, which were more marked in 0.8 µm pre-filtered treatment rather than in the whole community treatment, thus excluding the role of protistan grazers in our findings. Interestingly, a significant effect of temperature on reducing the average nucleic acid content (NAC) of prokaryotic cells in the communities was also observed. Cell size and nucleic acid decrease with temperature were correlated, showing a common mean decrease of 0.4% per °C. The usually larger HNA bacteria consistently showed a greater reduction in cell and NAC compared with their LNA counterparts, especially during the spring phytoplankton bloom period associated to maximum bacterial growth rates in response to nutrient availability. Our results show that the already smallest planktonic microbes, yet with key roles in global biogeochemical cycling, are likely undergoing important structural shrinkage in response to rising temperatures.

Temperature dependences of growth rates and carrying capacities of marine bacteria depart from metabolic theoretical predictions.

Using the metabolic theory of ecology (MTE) framework, we evaluated over a whole annual cycle the monthly responses to temperature of the growth rates (µ) and carrying capacities (K) of heterotrophic bacterioplankton at a temperate coastal site. We used experimental incubations spanning 6ºC with bacterial physiological groups identified by flow cytometry according to membrane integrity (live), nucleic acid content (HNA and LNA) and respiratory activity (CTC+). The temperature dependence of µ at the exponential phase of growth was summarized by the activation energy (E), which was variable (-0.52 to 0.72 eV) but followed a seasonal pattern, only reaching the hypothesized value for aerobic heterotrophs of 0.65 eV during the spring bloom for the most active bacterial groups (live, HNA, CTC+). K (i.e. maximum experimental abundance) peaked at 4 × 10(6) cells mL(-1) and generally covaried with µ but, contrary to MTE predictions, it did not decrease consistently with temperature. In the case of live cells, the responses of µ and K to temperature were positively correlated and related to seasonal changes in substrate availability, indicating that the responses of bacteria to warming are far from homogeneous and poorly explained by MTE at our site.

More, smaller bacteria in response to ocean's warming?

Heterotrophic bacteria play a major role in organic matter cycling in the ocean. Although the high abundances and relatively fast growth rates of coastal surface bacterioplankton make them suitable sentinels of global change, past analyses have largely overlooked this functional group. Here, time series analysis of a decade of monthly observations in temperate Atlantic coastal waters revealed strong seasonal patterns in the abundance, size and biomass of the ubiquitous flow-cytometric groups of low (LNA) and high nucleic acid (HNA) content bacteria. Over this relatively short period, we also found that bacterioplankton cells were significantly smaller, a trend that is consistent with the hypothesized temperature-driven decrease in body size. Although decadal cell shrinking was observed for both groups, it was only LNA cells that were strongly coherent, with ecological theories linking temperature, abundance and individual size on both the seasonal and interannual scale. We explain this finding because, relative to their HNA counterparts, marine LNA bacteria are less diverse, dominated by members of the SAR11 clade. Temperature manipulation experiments in 2012 confirmed a direct effect of warming on bacterial size. Concurrent with rising temperatures in spring, significant decadal trends of increasing standing stocks (3% per year) accompanied by decreasing mean cell size (-1% per year) suggest a major shift in community structure, with a larger contribution of LNA bacteria to total biomass. The increasing prevalence of these typically oligotrophic taxa may severely impact marine food webs and carbon fluxes by an overall decrease in the efficiency of the biological pump.

Polyunsaturated aldehydes from large phytoplankton of the Atlantic Ocean surface (42°n to 33°s).

Polyunsaturated aldehydes (PUAs) are organic compounds mainly produced by diatoms, after cell wounding. These compounds are increasingly reported as teratogenic for species of grazers and deleterious for phytoplanktonic species, but there is still scarce information regarding concentration ranges and the composition of PUAs in the open ocean. In this study, we analyzed the spatial distribution and the type of aldehydes produced by the large-sized (>10 µm) phytoplankton in the Atlantic Ocean surface. Analyses were conducted on PUAs released after mechanical disruption of the phytoplankton cells, referred to here as potential PUAs (pPUAs). Results show the ubiquitous presence of pPUA in the open ocean, including upwelling areas, as well as oligotrophic gyres. Total pPUA concentrations ranged from zero to 4.18 pmol from cells in 1 L. Identified PUAs were heptadienal, octadienal and decadienal, with heptadienal being the most common (79% of total stations). PUA amount and composition across the Atlantic Ocean was mainly related to the nitrogen:phosphorus ratio, suggesting nutrient-driven mechanisms of PUA production. Extending the range of trophic conditions considered by adding data reported for productive coastal waters, we found a pattern of PUA variation in relation to trophic status.