Metal stresses modify soluble proteomes and toxin profiles in two Mediterranean strains of the distributed dinoflagellate Alexandrium pacificum.

HABs involving Alexandrium pacificum have been reported in metal-contaminated ecosystems, suggesting that this distributed species adapts to and/or can tolerate the effects of metals. Modifications in soluble proteomes and PST contents were characterized in two Mediterranean A. pacificum strains exposed to mono- or polymetallic stresses (zinc, lead, copper, cadmium). These strains were isolated from two anthropized locations: Santa Giusta Lagoon (Italy, SG C10-3) and the Tarragona seaport (Spain, TAR C5-4F). In both strains, metals primarily downregulated key photosynthesis proteins. Metals also upregulated other proteins involved in photosynthesis (PCP in both strains), the oxidative stress response (HSP 60, proteasome and SOD in SG C10-3; HSP 70 in TAR C5-4F), energy metabolism (AdK in TAR C5-4F), neoglucogenesis/glycolysis (GAPDH and PEP synthase in SG C10-3) and protein modification (PP in TAR C5-4F). These proteins, possibly involved in adaptive proteomic responses, may explain the development of these A. pacificum strains in metal-contaminated ecosystems. The two strains showed different proteomic responses to metals, with SG C10-3 upregulating more proteins, particularly PCP. Among the PSTs, regardless of the metal and the strain studied, C2 and GTX4 predominated, followed by GTX5. Under the polymetallic cocktail, (i) total PSTs, C2 and GTX4 reached the highest levels in SG C10-3 only, and (ii) total PSTs, C2, GTX5 and neoSTX were higher in SG C10-3 than in TAR C5-4F, whereas in SG C10-3 under copper stress, total PSTs, GTX5, GTX1 and C1 were higher than in the controls, revealing variability in PST biosynthesis between the two strains. Total PSTs, C2, GTX4 and GTX1 showed significant positive correlations with PCP, indicating that PST production may be positively related to photosynthesis. Our results showed that the A. pacificum strains adapt their proteomic and physiological responses to metals, which may contribute to their ecological success in highly anthropized areas.

Intraspecific variability in membrane proteome, cell growth, and morphometry of the invasive marine neurotoxic dinoflagellate Alexandrium pacificum grown in metal-contaminated conditions.

Over the past decades, the occurrence, distribution and intensity of harmful algal blooms involving the dinoflagellate Alexandrium pacificum have increased in marine coastal areas disturbed by anthropogenic inputs. This invasive species produces saxitoxin, which causes the paralytic shellfish poisoning syndrome in humans upon consumption of contaminated seafood. Blooms of A. pacificum have been reported in metal-contaminated coastal ecosystems, suggesting some ability of these microorganisms to adapt to and/or resist in metal stress conditions. This study seeks to characterize the modifications in membrane proteomes (by 2-D electrophoresis coupled to LC-MS/MS), cell growth and morphometry (measured with an inverted microscope), in response to metal stress (addition of Zn2+, Pb2+, Cu2+ and Cd2+), in two Mediterranean A. pacificum strains: SG C10-3 and TAR C5-4F, respectively isolated from the Santa Giusta Lagoon (Sardinia, Italy) and from the Tarragona seaport (Spain), both metal-contaminated ecosystems. In the SG C10-3 cultures grown in a metal cocktail, cell growth was significantly delayed, and cell size increased (22% of 37.5 µm cells after 25 days of growth). Conversely, no substantial change was observed for cell growth or cell size in the TAR C5-4F cultures grown in a metal cocktail (P > 0.10), thus indicating intraspecific variability in the responses of A. pacificum strains to metal contamination. Regardless of the conditions tested, the total number of proteins constituting the membrane proteome was significantly higher for TAR C5-4F than for SG C10-3, which may help TAR C5-4F to thrive better in contaminated conditions. For both strains, the total number of proteins constituting the membrane proteomes was significantly lower in response to metal stress (29% decrease in the SG C10-3 proteome: 82 ± 12 proteins for controls, and 58 ± 12 in metal-contaminated cultures; 17% decrease in the TAR C5-4F proteome: 101 ± 8 proteins for controls, and 84 ± 5 in metal-contaminated cultures). Moreover, regardless of the strain, proteins with significantly modified expression in response to stress were mainly down-regulated (representing 45% of the proteome for SG C10-3 and 38% for TAR C5-4F), clearly showing the harmful effects of the metals. Protein down-regulation may affect cell transport (actin and phospholipid scramblase in SG C10-3), photosynthesis (RUBISCO in SG C10-3, light-harvesting protein in TAR C5-4F, and high-CO2-inducing periplasmic protein in both strains), and finally energy metabolism (ATP synthase in both strains). However, other modifications in protein expression may confer to these A. pacificum strains a capacity for adaptation and/or resistance to metal stress conditions, for example by (i) limiting the metal entry through the plasma membrane of the SG C10-3 cells (via the down-regulation of scramblase) and/or (ii) reducing the oxidative stress generated by metals in SG C10-3 and TAR C5-4F cells (due to down-regulation of ATP-synthase).

In silico prediction of the secretome from the invasive neurotoxic marine dinoflagellate Alexandrium catenella.

Alexandrium catenella, a marine dinoflagellate responsible for harmful algal blooms (HABs), proliferates with greater frequency, distribution and intensity, in disturbed marine coastal ecosystems. The proteins secreted into seawater may play a crucial role in maintaining this dinoflagellate in these ecosystems, but this possibility has never been investigated before. In this study, the A. catenella secretome was predicted from its transcriptome by combining several bioinformatics tools. Our results predict a secretome of 2 779 proteins, among which 79% contain less than 500 amino acids, suggesting that most secreted proteins are short in length. The predicted secretome includes 963 proteins (35%) with Pfam domains: 773 proteins with one Pfam domain and 190 proteins with two or more Pfam domains. Their functional annotation showed that they are mainly involved in (i) proteolysis, (ii) stress responses and (iii) primary metabolism. In addition, 47% of the secreted proteins appear to be enzymes, primarily peptidases, known to be biologically active in the extracellular medium during stress responses. Finally, this study provides a wealth of candidates of proteins secreted by A. catenella, which may interact with the marine environment and help this dinoflagellate develop in various environmental conditions.

Modifications of the soluble proteome of a mediterranean strain of the invasive neurotoxic dinoflagellate Alexandrium catenella under metal stress conditions.

The soluble proteome of the mediterranean strain ACT03 of the invasive neurotoxic dinoflagellate Alexandrium catenella exposed to lead or zinc at 6, 12 or 18µM (total concentrations), or under control conditions, was characterized by two-dimensional gel electrophoresis (2-DE). Zinc reduced (P<0.05) the total number of protein spots (-41%, -52% and -60%, at 6, 12 or 18µM, respectively). Besides, most of the proteins constituting the soluble proteome were down-regulated in response to lead or zinc stresses. These proteins were involved mainly in photosynthesis (20-37% for lead; 36-50% for zinc) (ribulose-1,5-bisphosphate carboxylase/oxygenase: RUBISCO; ferredoxin-NADP+ reductase: FNR; peridinin-chlorophyll a-protein: PCP), and in the oxidative stress response (29-34% for lead; 17-36% for zinc) (superoxide dismutase: SOD; proteasome α/ß subunits). These negative effects could be partly compensated by the up-regulation of specific proteins such as ATP-synthase ß subunit (+16.3 fold after exposure to lead at 12µM). Indeed, an increase in the abundance of ATP-synthase could enrich the ATP pool and provide more energy available for the cells to survive under metal stress, and make the ATP-synthase transport of metal cations out of the cells more efficient. Finally, this study shows that exposure to lead or zinc have a harmful effect on the soluble proteome of A. catenella ACT03, but also suggests the existence of an adaptative proteomic response to metal stresses, which could contribute to maintaining the development of this dinoflagellate in trace metal-contaminated ecosystems.

The marine bacteria Shewanella frigidimarina NCIMB400 upregulates the type VI secretion system during early biofilm formation.

Shewanella sp. are facultative anaerobic Gram-negative bacteria, extensively studied for their electron transfer ability. Shewanella frigidimarina has been detected and isolated from marine environments, and in particular, from biofilms. However, its ability to adhere to surfaces and form a biofilm is poorly understood. In this study, we show that the ability to adhere and to form a biofilm of S. frigidimarina NCIMB400 is significantly higher than that of Shewanella oneidensis in our conditions. We also show that this strain forms a biofilm in artificial seawater, whereas in Luria-Bertani, this capacity is reduced. To identify proteins involved in early biofilm formation, a proteomic analysis of sessile versus planktonic membrane-enriched fractions allowed the identification of several components of the same type VI secretion system gene cluster: putative Hcp1 and ImpB proteins as well as a forkhead-associated domain-containing protein. The upregulation of Hcp1 a marker of active translocation has been confirmed using quantitative reverse transcription polymerase chain reaction. Our data demonstrated the presence of a single and complete type VI secretion system in S. frigidimarina NCIMB400 genome, upregulated in sessile compared with planktonic conditions. The fact that three proteins including the secreted protein Hcp1 have been identified may suggest that this type VI secretion system is functional.

Three-dimensional (3-D) fluorescence spectroscopy analysis of the fluorescent dissolved organic matter released by the marine toxic dinoflagellate Alexandrium catenella exposed to metal stress by zinc or lead.

We investigated the effects of zinc or lead on growth and on exudation of fluorescent dissolved organic matter (FDOM) by the marine toxic dinoflagellate Alexandrium catenella (Whedon & Kofoid) Balech. The species was exposed to increasing free zinc (1.34 × 10(-7) M-3.98 × 10(-6) M) or lead (5.13 × 10(-9) M-1.82 × 10(-7) M) concentra-tions. Low metal levels ([Zn(2+) ] = 1.34 × 10(-7) M; [Pb(2+) ] = 5.13 × 10(-9) M) had no effect on cell growth. Toxic effects were observed from higher metal contamination ([Zn(2+) ] = 3.98 × 10(-6) M; [Pb(2+) ] = 6.54 × 10(-8) M), as a conversion of vegetative cells into cysts. Analysis of the released FDOM by three-dimensional (3-D) fluorescence spectroscopy was achieved, using the parallel factor analysis (PARAFAC). The PARAFAC modeling revealed four components associated with two contributions: one related to the biological activity; the other linked to the organic matter decomposition in the culture medium. The C1 component combined a tryptophan peak and characteristics of humic substances, whereas the C2 component was considered as a tryptophan protein fluorophore. The two others C3 and C4 components were associated with marine organic matter production. Relea-sed fluorescent substances were induced by low ([Zn(2+) ]= 1.34 × 10(-7) M; [Pb(2+) ] = 5.13 × 10(-9) M) and moderate ([Zn(2+) ] = 6.21 × 10(-7) M; [Pb(2+) ] = 2.64× 10(-9) M) metal concentrations, suggesting the activation of cellular mechanisms in response to metal stress, to exudate FDOM that could complex metal cations and reduce their toxicity toward A. catenella cells.

Copper and cadmium effects on growth and extracellular exudation of the marine toxic dinoflagellate Alexandrium catenella: 3D-fluorescence spectroscopy approach.

In this study, metal contamination experiments were conducted to investigate the effects of copper and cadmium on the growth of the marine toxic dinoflagellate Alexandrium catenella and on the production of dissolved organic matter (Dissolved Organic Carbon: DOC; Fluorescent Dissolved Organic Matter: FDOM). This species was exposed to increasing concentrations of Cu(2+) (9.93 × 10(-10)-1.00 × 10(-7)M) or Cd(2+) (1.30 × 10(-8)-4.38 × 10(-7)M), to simulate polluted environments. The drastic effects were observed at pCu(2+)=7.96 (Cu(2+): 1.08 × 10(-8)M) and pCd(2+)=7.28 (Cd(2+): 5.19 × 10(-8)M), where cyst formation occurred. Lower levels of Cu(2+) (pCu(2+)>9.00) and Cd(2+) (pCd(2+)>7.28) had no effect on growth. However, when levels of Cu(2+) and Cd(2+) were beyond 10(-7)M, the growth was totally inhibited. The DOC released per cell (DOC/Cell) was different depending on the exposure time and the metal contamination, with higher DOC/Cell values in response to Cu(2+) and Cd(2+), comparatively to the control. Samples were also analyzed by 3D-fluorescence spectroscopy, using the Parallel Factor Analysis (PARAFAC) algorithm to characterize the FDOM. The PARAFAC analytical treatment revealed four components (C1, C2, C3 and C4) that could be associated with two contributions: one, related to the biological activity; the other, linked to the decomposition of organic matter. The C1 component combined a tryptophan peak and a characteristic humic substances response, and the C2 component was considered as a tryptophan protein fluorophore. The C3 and C4 components were associated to marine organic matter production.

Protein expression from zooplankton communities in a metal contaminated NW mediterranean coastal ecosystem.

Bidimensional and monodimensional polyacrylamide gel electrophoresis were used to study protein expression from zooplankton collected in thirteen stations of Toulon Bay (NW Mediterranean). In this ecosystem, Little Bay showed higher trace metal concentrations (13.5-23.8 nM for Cu, 0.73-1.24 nM for Pb, 27.8-58.7 nM for Zn) than Large Bay (Cu 2.2-15.6 nM; Pb 0.19-0.78 nM; Zn 9.0-38.8 nM). Trace metals positively correlated (p < 0.05) with expression of four zooplankton proteins (MW in kDa/pI: 25.0/5.6; 48.8/4.1; 38.2/4.4; 38.3/5.8) and with biomass of Oithona nana, predominant copepod in Little Bay. Sequencing by LC-MS/MS putatively provided zooplankton identity of these proteins: they were cytoskeleton actin, except one protein that was the chaperone calreticulin. We suggest that actin and calreticulin could be regarded as zooplankton markers of metal stress and be involved in a possible tolerance of O. nana to contamination, contributing to its development in a marine perturbed ecosystem.

Seasonal changes in phosphatase activities in Toulon Bay (France).

We studied the characteristics of the phosphatase activity (Km and Vmax) in total seawater and in particulate material of the three main plankton classes (0.25-5, 5-90 and >90 microm) in a coastal marine ecosystem of Toulon Bay (French Mediterranean Sea). The measurement of the hydrolysis of sodium paranitrophenylphosphate (pNPP), a substrate of phosphatase, revealed low and high affinity components in unfiltered seawater and in particulate matter. In unfiltered seawater, the low affinity activity was predominant from October to March during phytoplankton development. The high affinity activity dominated from April to June and was significantly correlated with the bacterial abundances. The phosphatase behaviour in the particulate material differs from that in the unfiltered seawater. The activity of the three particulate classes was generally much lower than that of unfiltered seawater, particularly the low affinity activity. The >90 microm size fraction consisted in greater part of zooplankton. In this size class, the activity (nmol l(-1) h(-1)) of the low affinity component was predominant from May to August, when the abundance of the larvae of copepods (copepodites) was highest. Its high specific activity (Activity/Protein concentration as nmol l(-1) h(-1) microg(-1)) was particularly elevated during this period. The 5-90 microm fraction consisted of phytoplankton cells, especially Dinoflagellates. Between September and January, the activity (nmol l(-1) h(-1)) of this size class was mostly supported by the low affinity component. The specific activity (nmol l(-1) h(-1) microg(-1)) of the high affinity component was highest in June and August. No significant correlation was found between phosphatase activities and chlorophyll a or total cell abundance. In return temporary relationships with specific taxa exist in particular with Ceratium spp., Gymnodinium spp. and Protoperidinium spp. The contribution of the 0.25-5 microm size class exceeded rarely 20% of the total particulate activity. Between June and August, high specific activities (nmol l(-1) h(-1) microg(-1)) were observed for its high affinity component. In autumn, strong rainfall increased the phosphate and nitrate concentrations and led to a drop in salinity, which probably explains the low phosphatase activities (nmol l(-1) h(-1)) and cell densities observed during this period.

Seasonal changes in zooplanktonic alkaline phosphatase activity in Toulon Bay (France): the role of Cypris larvae.

We studied zooplankton contribution to the total particulate phosphatase activity, the kinetics of this activity, the relation to the different taxonomic groups and the role of particle-bound bacteria. The activity of total particulate material collected from a liter of seawater was more elevated in May, June and August than during the rest of the year. These high activities resulted from a high contribution of the >90 microm fraction which account then for more than 60% of the total particulate activity. Two Michaelian processes with high and low V(max) were disclosed on this fraction. The high V(max) component was responsible for the high summer activities. During these periods, high densities of cirriped Cypris were found which were statistically correlated with this high V(max) component as with its specific activity. Moreover, the contribution of attached bacteria to these high activities was low. In return, this contribution was predominant during the periods of low activity. A simple method was developed to characterise this bacterial activity.