Variable inter and intraspecies alkaline phosphatase activity within single cells of revived dinoflagellates

Adaptation of cell populations to environmental changes is mediated by phenotypic variability at the single-cell level. Enzyme activity is a key factor in cell phenotype and the expression of the alkaline phosphatase activity (APA) is a fundamental phytoplankton strategy for maintaining growth under phosphate-limited conditions. Our aim was to compare the APA among cells and species revived from sediments of the Bay of Brest (Brittany, France), corresponding to a pre-eutrophication period (1940’s) and a beginning of a post-eutrophication period (1990’s) during which phosphate concentrations have undergone substantial variations. Both toxic marine dinoflagellate Alexandrium minutum and the non-toxic dinoflagellate Scrippsiella acuminata were revived from ancient sediments. Using microfluidics, we measured the kinetics of APA at the single-cell level. Our results indicate that all S. acuminata strains had significantly higher APA than A. minutum strains. For both species, the APA in the 1990’s decade was significantly lower than in the 1940’s. For the first time, our results reveal both inter and intraspecific variabilities of dinoflagellate APA and suggest that, at a half-century timescale, two different species of dinoflagellate may have undergone similar adaptative evolution to face environmental changes and acquire ecological advantages.Subject terms: Climate-change ecology, Microbial ecology     

The role of phosphorus on planktonic production of the Gironde plume waters in the Bay of Biscay

More and more studies emphasize the status of phosphorus (P)as the principal limiting nutrient of phytoplankton growth,especially in coastal waters under the influence of freshwaterdischarges. The purpose of the present paper is to investigatethe role of P on planktonic production in the waters influencedby the Gironde discharges; the Gironde being one of the twolargest rivers on the French Atlantic coast. The survey is basedon several cruises made in 1998 and 1999. Two different patternswere observed for waters with salinity below and above 34.5.For waters with salinity < 34.5, P was found to be the firstlimiting nutrient of winter and spring phytoplankton blooms,based on undetectable phosphate (< 20 nM), high NO₃ : PO₄ratios, typically > 100 : 1, short phosphate turnover time(1 to 2 h), high alkaline phosphatase activities (mean of 176nM h^-1 in late May 1999) and ultimately great increases of chlorophylla (Chl a) and primary production in phosphate-enriched samplesrelative to controls. This limitation could be partly explainedby the Gironde nutrient supplies, which were phosphate deficientcompared with the mineral nitrogen(N_min : PO₄ was > 40 withina salinity range 16–33). In summer, corresponding to theperiod of low influence of Gironde supplies (low runoff anda spreading effect of the plume), phytoplankton growth wouldbe controlled by both P and nitrogen (N), according to low nitrateand the major effect of combined P+N (NH₄) enrichment on Chla and primary production compared with the addition of N orP singly. In early October, after the first autumn gales, themixed layer was enriched with a sufficient supply of nutrientsto support exponential phytoplankton growth for 4 days in enclosures.The pattern was different for waters at the limit of the Girondeplume and Atlantic oceanic waters (within salinity range 34.5–35.4).P would not be the single limiting nutrient of winter bloomsand spring phytoplankton growth since low phosphate, and alsolow nitrate and silicate, availability were recorded and phosphateaddition alone had no effect on phytoplankton biomass and productionin bioassays. The early P limitation of winter blooms had consequencesfor the phytoplankton community structure in the Gironde plumewaters (salinity < 34.5). Whereas major cells of these bloomswere greater than 20 µm in size, phytoplankton growthin spring and autumn was dominated by 3–20 µm (e.g.53% of Chl a in late April 1999) and < 3 µm cells (e.g.29% of Chl a). The decreasing size of phytoplankton cells isemphasized by the severe competition between bacteria and algaefor phosphate, since bacteria dominated phosphate uptake inspring (e.g. 87% in late April, 77% in late May). Bacteria tendedto have greater affinity for phosphate and seemed also to beP limited at certain times in spring, according to results fromphosphate enrichment bioassays in late May 1999. The alternativemethod for phytoplankton to obtain P would be the use of thedissolved organic phosphorus pool by alkaline phosphatase activity.According to the movement of ³³P after initial labelling ofmicrobial populations and a subsequent cold chase, the majortransfer of P occurred from the bacterial to the dissolved fraction.We hypothesize that algae obtain part of its dissolved organicphosphorus from bacteria-originated organic phosphorus compounds.     

GROWTH AND PHOSPHORUS UPTAKE BY THE TOXIC DINOFLAGELLATE ALEXANDRIUM CATENELLA (DINOPHYCEAE) IN RESPONSE TO PHOSPHATE LIMITATION1

Alexandrium catenella (Whedon et Kof.) Balech has exhibited seasonal recurrent blooms in the Thau lagoon (South of France) since first reported in 1995. Its appearance followed a strong decrease (90%) in phosphate (PO₄^3?) concentrations in this environment over the 1970–1995 period. To determine if this dinoflagellate species has a competitive advantage in PO₄^3?‐limited conditions in terms of nutrient acquisition, semicontinuous cultures were carried out to characterize phosphorus (P) uptake by A. catenella cells along a P‐limitation gradient using different dilution rates (DRs). Use of both inorganic and organic P was investigated from measurements of ³³PO₄^3? uptake and alkaline phosphatase activity (APA), respectively. P status was estimated from cellular P and carbon contents (Q_P and Q_C). Shifts in trends of Q_P/Q_C and Q_P per cell (Q_P·cell?1) along the DR gradient allowed the definition of successive P‐stress thresholds for A. catenella cells. The maximal uptake rate of ³³PO₄^3? increased strongly with the decrease in DR and the decrease in Q_P/Q_C, displaying physiological acclimations to PO₄^3? limitation. Concerning maximal APA per cell, the observation of an all‐or‐nothing pattern along the dilution gradient suggests that synthesis of AP was induced and maximized at the cellular scale as soon as PO₄^3? limitation set in. APA variations revealed that the synthesis of AP was repressed over a PO₄^3? threshold between 0.4 and 1 μM. As lower PO₄^3? concentrations are regularly observed during A. catenella blooms in Thau lagoon, a significant portion of P uptake by A. catenella cells in the field may come from organic compounds.     

NITROGEN UPTAKE AND ASSIMILATION KINETICS IN ALEXANDRIUM MINUTUM (DYNOPHYCEAE): EFFECT OF N‐LIMITED GROWTH RATE ON NITRATE AND AMMONIUM INTERACTIONS1

Uptake and assimilation kinetics of nitrate and ammonium were investigated along with inhibition of nitrate uptake by ammonium in the harmful dinoflagellate Alexandrium minutum Halim at different nitrogen (N)–limited growth rates. Alexandrium minutum had a strong affinity for nitrate and ammonium (K_s=0.26±0.03 and 0.31±0.04 μmol·L^?1, respectively) whatever the degree of N deficiency of the cells. Ammonium was always the preferred form of nitrogen taken up (=0.42–0.50). In the presence of both forms, nitrate uptake was inhibited by ammonium, and inhibition was particularly marked in N‐sufficient cells (I_max～0.9 and K_i=0.31–0.56 μmol·L^?1). In the case of N assimilation, ammonium was also the preferred form in N‐deficient cells (=0.54–0.72), whereas in N‐sufficient cells, both N sources were equally preferred (=0.90–1.00). The comparison of uptake and assimilation rates highlighted the ability of A. minutum to significantly store in 1 h nitrate and ammonium in amounts sufficient to supply twice the daily N requirements of the slowest‐growing N‐deficient cells. Nitrogen uptake kinetic parameters of A. minutum and their ecological implications are discussed.     

Quorum sensing disruption regulates hydrolytic enzyme and biofilm production in estuarine bacteria

Biofilms of heterotrophic bacteria cover organic matter aggregates and constitute hotspots of mineralization, primarily acting through extracellular hydrolytic enzyme production. Nevertheless, regulation of both biofilm and hydrolytic enzyme synthesis remains poorly investigated, especially in estuarine ecosystems. In this study, various bioassays, mass spectrometry and genomics approaches were combined to test the possible involvement of quorum sensing (QS) in these mechanisms. QS is a bacterial cell–cell communication system that relies notably on the emission of N-acylhomoserine lactones (AHLs). In our estuarine bacterial collection, we found that 28 strains (9%), mainly Vibrio, Pseudomonas and Acinetobacter isolates, produced at least 14 different types of AHLs encoded by various luxI genes. We then inhibited the AHL QS circuits of those 28 strains using a broad-spectrum lactonase preparation and tested whether biofilm production as well as β-glucosidase and leucine-aminopeptidase activities were impacted. Interestingly, we recorded contrasted responses, as biofilm production, dissolved and cell-bound β-glucosidase and leucine-aminopeptidase activities significantly increased in 4%–68% of strains but decreased in 0%–21% of strains. These findings highlight the key role of AHL-based QS in estuarine bacterial physiology and ultimately on biogeochemical cycles. They also point out the complexity of QS regulations within natural microbial assemblages.     

Phytoplankton and bacterial alkaline phosphatase activities in relation to phosphate and DOP availability within the Gironde plume waters (Bay of Biscay)

Previous studies conducted on the continental shelf in the Southeast Bay of Biscay influenced by Gironde waters (one of the two largest rivers on the French Atlantic coast) showed the occurrence of late winter phytoplankton blooms and phosphorus limitation of algal growth thereafter. In this context, the importance of dissolved organic phosphorus (DOP) for both algae and bacteria was investigated in 1998 and 1999 in terms of stocks and fluxes. Within the mixed layer, although phosphate decreased until exhaustion from winter to spring, DOP remained high and phosphate monoesters made up between 11 to 65% of this pool. Total alkaline phosphatase activity (APA, V_max) rose gradually from winter (2-8 nM h⁻¹) to late spring (100-400 nM h⁻¹), which was mainly due to an increase in specific phytoplankton (from 0.02 to 3.0 nmol μgC⁻¹ h⁻¹) and bacterial APA (from 0.04 to 4.0 nmol μgC⁻¹ h⁻¹), a strategy to compensate for the lack of phosphate. At each season, both communities had equal competitive abilities to exploit DOP but, taking into account biomass, the phytoplankton community activity always dominated (57-63% of total APA) that of bacterial community (9-11%). The dissolved APA represented a significant contribution. In situ regulation of phytoplanktonic APA by phosphate (induction or inversely repression of enzyme synthesis) was confirmed by simultaneously conducted phosphate-enrichment bioassays. Such changes recorded at a time scale of a few days could partly explain the seasonal response of phytoplankton communities to phosphate depletion.