Detection of Extracellular Phosphatase Activity of Heterotrophic Prokaryotes at the Single‐Cell Level by Flow Cytometry

Monitoring cell activity using substrates, which turn fluorescent due to biological activity, allows observing the presence and dynamics of sub‐populations, and provides a very valuable insight in ecological studies. The phosphatase substrate ELF97 phosphate (ELF‐P) is a useful tool to detect and quantify phosphatase activity (PA) of microorganisms at the single‐cell level. Most of the studies dealing with PA at the single‐cell level focus on autotrophic cells and only few concern heterotrophic prokaryotes (referred as bacteria in the text). While flow cytometry is a promising tool to assess the single‐cell analysis, only microscopy tools have been used until now to measure the ELF labeling associated with bacteria expressing PA. Therefore, we have developed a new protocol that enables the detection of ELF alcohol (ELFA), the product of ELF‐P hydrolysis, making possible the specific identification of bacteria showing PA using flow cytometry

Analysis of Phosphatase Activity from Aquatic Heterotrophic Bacteria at the Single Cell Level by Flow Cytometry: Example of a Development Achieved in the Regional Flow Cytometry Platform for Microbiology (Precym) Hosted by the Oceanology Center of Marseille

Marine microbes play a very important role in biogeochemical cycles. Because of their ­biodiversity, the diversity of their metabolisms, and their ­physiological heterogeneity (live/dead, active/inactive cells), it is necessary to study them at the single cell level. Flow cytometry has become a technique of choice to reach this goal, providing a fast, reliable, and multiparametric analysis of individual cells. However, the cost of such a technique makes flow cytometry out of reach for small institutes or laboratory units. It also requires a qualified and well-trained operator. To make this technique available to all the microbiologists of the “Provence Alpes Côte d’Azur” Region, a Regional Flow Cytometry Platform (PRECYM), hosted by the Oceanology Centre of Marseille, was created by a consortium of six laboratories. Its goal is to provide researchers with the possibility to consider new approaches and to guarantee optimal technical, economic, and scientific exploitations of these instruments. An example of such a technical development is described in this chapter. The ELF97 phosphatase substrate has been shown to be a performing tool to assess the phosphorus status of phytoplankton populations. Recently, this technique has been successfully applied to marine heterotrophic bacteria in culture samples in which ELF-labeling was observed by microscopy. In this study, we describe a new protocol for ­ELF-labeling of heterotrophic bacteria that allow detection by flow cytometry. ELF-labeled samples were stored in liquid nitrogen for up to 4 months before analysis without any significant loss of total or ELF-labeled cell abundance and of relative ELF ­fluorescence intensity. This method enables studying ectoenzymatic alkaline phosphatase activity of heterotrophic bacteria at the single cell level in environments even with low cell abundance. It has been successfully applied in both fresh and marine samples. This approach is set to improve our understanding of the physiological response of heterotrophic bacteria to phosphorus limitation

Phospholipid synthesis rates in the eastern subtropical South Pacific Ocean

Membrane lipid molecules are a major component of planktonic organisms and this is particularly true of the microbial picoplankton that dominate the open ocean; with their high surface-area to volume ratios, the synthesis of membrane lipids places a major demand on their overall cell metabolism. Specifically, the synthesis of cell membrane phospholipids creates a demand for the nutrient phosphorus, and we sought to refine our understanding of the role of phospholipids in the upper ocean phosphorus cycle. We measured the rates of phospholipid synthesis in a transect of the eastern subtropical South Pacific from Easter Island to Concepcion, Chile as part of the BIOSOPE program. Our approach combined standard phosphorus radiotracer incubations and lipid extraction methods. We found that phospholipid synthesis rates varied from less than 1 to greater than 200 pmol P L−1 h−1, and that phospholipid synthesis contributed between less than 5% to greater than 22% of the total PO43− incorporation rate. Changes in the percentage that phospholipid synthesis contributed to total PO43− uptake were strongly correlated with the ratio of primary production to bacterial production, which supported our hypothesis that heterotrophic bacteria were the primary agents of phospholipid synthesis. The spatial variation in phospholipid synthesis rates underscored the importance of heterotrophic bacteria in the phosphorus cycle of the eastern subtropical South Pacific, particularly the hyperoligotrophic South Pacific subtropical gyre

Heterotrophic bacterial production in the eastern South Pacific: longitudinal trends and coupling with primary production

Spatial variation of heterotrophic bacterial production and phytoplankton primary production were investigated across the eastern South Pacific Ocean (−141° W, −8° S to −72° W, −35° S) in November–December 2004. Bacterial production (³H leucine incorporation) integrated over the euphotic zone encompassed a wide range of values, from 43 mg C m⁻² d⁻¹ in the hyper-oligotrophic South Pacific Gyre to 392 mg C m⁻² d⁻¹ in the upwelling off Chile. In the gyre (120° W, 22° S) records of low phytoplankton biomass (7 mg Total Chla m⁻²) were obtained and fluxes of in situ 14C-based particulate primary production were as low as 153 mg C m⁻² d⁻¹, thus equal to the value considered as a limit for primary production under strong oligotrophic conditions. Average rates of ³H leucine incorporation rates, and leucine incorporation rates per cell (5–21 pmol l⁻¹ h⁻¹ and 15–56×10⁻²¹ mol cell⁻¹ h⁻¹, respectively) determined in the South Pacific gyre, were in the same range as those reported for other oligotrophic subtropical and temperate waters. Fluxes of dark community respiration, determined at selected stations across the transect varied in a narrow range (42–97 mmol O2 m⁻² d⁻¹), except for one station in the upwelling off Chile (245 mmol O2 m⁻² d⁻¹). Bacterial growth efficiencies varied between 5 and 38%. Bacterial carbon demand largely exceeded 14C particulate primary production across the South Pacific Ocean, but was lower or equal to gross community production

Dissolved organic matter stimulates N₂ fixation and nifH gene expression in Trichodesmium

Mixotrophy, the combination of heterotrophic and autotrophic nutrition modes, is emerging as the rule rather than the exception in marine photosynthetic plankton. Trichodesmium, a prominent diazotroph ubiquitous in the (sub)tropical oceans, is generally considered to obtain energy via autotrophy. While the ability of Trichodesmium to use dissolved organic phosphorus when deprived of inorganic phosphorus sources is well known, the extent to which this important cyanobacterium may benefit from other dissolved organic matter (DOM) sources is unknown. Here we provide evidence of carbon-, nitrogen- and phosphorus-rich DOM molecules enhancing N₂ fixation rates and nifH gene expression in natural Trichodesmium colonies collected at two stations in the western tropical South Pacific. Sampling at a third station located in the oligotrophic South Pacific Gyre revealed no Trichodesmium but showed presence of UCYN-B, although no nifH expression was detected. Our results suggest that Trichodesmium may behave mixotrophically in response to certain environmental conditions, providing them with metabolic plasticity and adding up to the view that mixotrophy is widespread among marine microbes

Nutrients limitation of primary productivity in the Southeast Pacific (BIOSOPE cruise)

Revue sans Comité de lectureInternational audienceIron is an essential nutrient involved in a variety of biological processes in the ocean, including photosynthesis, respiration and nitrogen fixation. Atmospheric deposition of aerosols is recognized as the main source of iron for the surface ocean. In high nutrient, low chlorophyll areas, it is now clearly established that iron limits phytoplankton productivity but its biogeochemical role in low nutrient, low chlorophyll environments has been poorly studied. We investigated this question in the unexplored southeast Pacific, arguably the most oligotrophic area of the global ocean. Situated far from any continental aerosol source, the atmospheric iron flux to this province is amongst the lowest of the world ocean. Here we report that, despite low dissolved iron concentrations (~0.1 nmol l-1) measured across the whole gyre (3 stations situated in the center, the western and the eastern edge), photosynthesis and primary productivity are only limited by iron availability at the border of the gyre, but not in the center. The seasonal stability of the gyre has apparently allowed for the development of populations acclimated to these extreme oligotrophic conditions. Moreover, despite clear evidence of nitrogen limitation in the central gyre, we were unable to measure nitrogen fixation in our experiments, even after iron and/or phosphate additions, and cyanobacterial nifH gene abundances were extremely low compared to the North Pacific Gyre. The South Pacific gyre is therefore unique with respect to the physiological status of its phytoplankton populations

Deep maxima of phytoplankton biomass, primary production and bacterial production in the Mediterranean Sea

The deep chlorophyll maximum (DCM) is a ubiquitous feature of phytoplankton vertical distribution in stratified waters that is relevant to our understanding of the mechanisms that underpin the variability in photoautotroph ecophysiology across environmental gradients and has implications for remote sensing of aquatic productivity. During the PEACETIME (Process studies at the air-sea interface after dust deposition in the Mediterranean Sea) cruise, carried out from 10 May to 11 June 2017, we obtained 23 concurrent vertical profiles of phytoplankton chlorophyll a, carbon biomass and primary production, as well as heterotrophic prokaryotic production, in the western and central Mediterranean basins. Our main aims were to quantify the relative role of photoacclimation and enhanced growth as underlying mechanisms of the DCM and to assess the trophic coupling between phytoplankton and heterotrophic prokaryotic production. We found that the DCM coincided with a maximum in both the biomass and primary production but not in the growth rate of phytoplankton, which averaged 0.3 d−1 and was relatively constant across the euphotic layer. Photoacclimation explained most of the increased chlorophyll a at the DCM, as the ratio of carbon to chlorophyll a (C : Chl a) decreased from ca. 90–100 (g : g) at the surface to 20–30 at the base of the euphotic layer, while phytoplankton carbon biomass increased from ca. 6 mgCm−3 at the surface to 10–15 mgCm−3 at the DCM. As a result of photoacclimation, there was an uncoupling between chlorophyll a-specific and carbon-specific productivity across the euphotic layer. The ratio of fucoxanthin to total chlorophyll a increased markedly with depth, suggesting an increased contribution of diatoms at the DCM. The increased biomass and carbon fixation at the base of the euphotic zone was associated with enhanced rates of heterotrophic prokaryotic activity, which also showed a surface peak linked with warmer temperatures. Considering the phytoplankton biomass and turnover rates measured at the DCM, nutrient diffusive fluxes across the nutricline were able to supply only a minor fraction of the photoautotroph nitrogen and phosphorus requirements. Thus the deep maxima in biomass and primary production were not fuelled by new nutrients but likely resulted from cell sinking from the upper layers in combination with the high photosynthetic efficiency of a diatom-rich, low-light acclimated community largely sustained by regenerated nutrients. Further studies with increased temporal and spatial resolution will be required to ascertain if the peaks of deep primary production associated with the DCM persist across the western and central Mediterranean Sea throughout the stratification season

Fate of Phaeodactylum tricornutum and nitrogen flow in an experimental microbial food web limited at the top by protozoans

International audienceThe fate of phytoplankton nitrogen through a simplified microbial food web was investigated. In a 2-stage culture system, I separated production and degradation processes of a diatom, Phaeodactylum tricornutum. In the first stage, the algae grew axenically and continuously. In the second stage, maintained in the dark, a 20 pm ciliate grazed on the diatoms and constituted the last trophic step. A microbial loop, based on bacterial utilization of partially degraded P. tricornutum cells, egested with the clliate digestive vacuoles, developed rapidly. C h a t e production was maintained only because the P. tricornutum grazer was able to feed simultaneously on bacterivorous nanoflagellates, themselves preying upon bacteria using egested material of the chate. Dissolved organic nitrogen rapidly constituted more than 50% of the identified nitrogen forms (&ssolved + particulate; mineral + organic). The nitrogen budget compiled during the succession of the different prey-predator relationships revealed the important role of dissolved organic matter adsorption on particles and aggregate formation including unstable organic colloids. The temporary importance and turnover of colloidal nitrogen was thus demonstrated

Influence of Phytoplankton Lysis or Grazing on Bacterial Metabolism and Trophic Relationships

International audienceExperimental microcosms were used to study the dynamics of heterotrophic bacterial populations with respect to phytoplankton loss. In a two-stage linked culture system, we artificially separated production and loss processes of a diatom Phaeodactylum tricornutum. In the first (productive) stage, the algae developed axenically and continuously. The outfow was fluxed in two degradation stages, where phytoplankton-derived detritus re-suited respectively from: (1) excretion and by-products of phagotrophic organisms (protozoans), and (2) bacterial degradation through bacterial attachment and lysis. According to the phytoplankton decay mode, i.e., lysis or grazing, bacterial adaptations were different. The study of bacterial productivity and aminopeptidase activity showed specific bacterial evolution during the succession of different prey-predator relationships. The occurrence of aggregates allowed nanoflagellates to develop an alternative diet; they fed not only on bacteria, but also on partially degraded phytoplankton detritus, inducing a strong shortcut in the food chain. Sources and controls of extracellular prote-olytic activity are discussed. Such experimental approaches are interesting because they separate bacterial lysis and protozoan grazing of phytoplankton, as well as the fates of their corresponding phytoplankton detritus in the microbial food web