Assessment of Metacaspase Activity in Phytoplankton.

Programmed cell death (PCD) is an irreversible, genetically-controlled form of cell suicide in which an endogenous biochemical pathway leads to morphological changes and ultimately, cellular demise. PCD is accompanied by de-novo protein synthesis of a family of proteases-"caspases" that are often used as a diagnostic marker of PCD. Although phytoplankton do not contain true caspases, caspase-like activity (hypothetical proteins with analogous activity) has been traditionally used as a diagnostic marker of PCD in marine phytoplankton. Increased caspase-like proteolytic activity was demonstrated when synthetic fluorogenic activity substrates specific for caspases (with an Asp at the P1 position) were applied upon PCD induction. Metacaspases, cysteine proteases, share structural properties with those of caspases, yet they are highly specific for Arg and Lys cleavage site at the P1 position implying that caspase specific substrates are not indicative of metacaspase catalytic activity. This method specifically tests direct metacaspase activity in phytoplankton by the cleavage of the fluorogenic metacaspase substrate Ac-VRPR-AMC. Metacaspase activity was tested by the addition of a metacaspase specific peptide that is conjugated to the fluorescent reporter molecule. The cleavage of the peptide by the metacaspase releases the fluorochrome that, when excited by light, emits fluorescence. The level of metacaspase enzymatic activity in the cell lysate is directly proportional to the fluorescence signal detected. The use of specific standards in this test enables the quantification of the fluorescence results. This assay directly allows monitoring the metacaspase cleavage products and thereby tracing evidence for programmed cell death.

Metacaspase involvement in programmed cell death of the marine cyanobacterium Trichodesmium.

Metacaspases are cysteine specific proteases implicated in cell-signalling, stress acclimation and programmed cell death (PCD) pathways in plants, fungi, protozoa, bacteria and algae. We investigated metacaspase-like gene expression and biochemical activity in the bloom-forming, N2 -fixing, marine cyanobacterium Trichodesmium, which undergoes PCD under low iron and high-light stress. We examined these patterns with respect to in-silico analyses of protein domain architectures that revealed a diverse array of regulatory domains within Trichodesmium metacaspases-like (TeMC) proteins. Experimental manipulations of laboratory cultures and oceanic surface blooms of Trichodesmium from the South Pacific Ocean triggered PCD under Fe-limitation and high light along with enhanced TeMC activity and upregulated expression of diverse TeMC representatives containing different regulatory domains. Furthermore, TeMC activity was significantly and positively correlated with caspase-like activity, which has been routinely observed to increase with PCD induction in Trichodesmium. Although both TeMC and caspase-like activities were stimulated upon PCD induction, inhibitor treatments of these proteolytic activities provided further evidence of largely distinct substrate specificities, even though some inhibitory crossover was observed. Our findings are the first results linking metacaspase expression and activity in PCD induced mortality in Trichodesmium. Yet, the role/s and specific activities of these different proteins remain to be elucidated.

Iron and phosphorus deprivation induce sociality in the marine bloom-forming cyanobacterium Trichodesmium.

Trichodesmium spp. are diazotrophic cyanobacteria that exist as single filaments (trichomes) and as macroscopic colonies of varying shapes formed by aggregating trichomes. The causes and dynamics of colony formation and disassociation are not yet elucidated. we demonstrate that limited availability of dissolved phosphorus (P) or iron (Fe) stimulated trichome mobility and induced colony formation in Trichodesmium erythraeum IMS101 cultures. The specific nutrient limitation differentially affected the rate of colony formation and morphology of the colonies. Fe starvation promoted rapid colony formation (10-48 h from depletion) while 5-7 days were required for colonies to form in P-depleted cultures. Video analyses confirmed that the probability of trichomes to cluster increased from 12 to 35% when transferred from nutrient replete to Fe-depleted conditions. Moreover, the probability for Fe-depleted aggregates to remain colonial increased to 50% from only 10% in nutrient replete cultures. These colonies were also characterized by stronger attachment forces between the trichomes. Enrichment of nutrient-depleted cultures with the limited nutrient-stimulated colony dissociation into single trichomes. We postulate that limited P and Fe availability enhance colony formation of Trichodesmium and primarily control the abundance and distribution of its different morphologies in the nutrient-limited surface ocean.

The effect of coagulants and antiscalants discharged with seawater desalination brines on coastal microbial communities: A laboratory and in situ study from the southeastern Mediterranean.

Desalination outflows frequently discharge brine containing coagulants and antiscalants (e.g. Iron-hydroxides and polyphosphonates) to the coastal environment. Here we examined changes in composition and productivity of natural microbial coastal communities in experimental mesocosms treated with either iron-hydroxide (Fe), polyphosphonate (Pn), or a combination of high salinities with both chemicals (All). Within 2 h of addition Fe already altered the microbial community composition, enhanced the bacterial production (BP) and cell specific production (BP/BA), and decreased primary production. Addition of Pn, relieved phosphorus stress as demonstrated by the immediate (within 2 h) and significant reduction in the ecto-enzyme alkaline phosphatase activity (APA). Synergistic effects were observed in the All treatment, reflected by increased production of both primary and bacterial producers as P-stress was relieved. After 10 days of incubation, the microbial community composition changed significantly only in the All treatment. The Fe-only treatment caused a significant decline in autotrophic biomass and in the assimilation number (AN), while in both the Pn and the All treatments the BP/BA increased with the added P. We also examined the microbial community responses in a natural impacted environment at the Ashkelon seawater desalination plant brine discharge site during summer and winter. The community composition differed in elevated-salinity compared with non-impacted stations with higher AN and bacterial efficiencies (BP/BA) measured in summer in the elevated-salinity stations. The seasonal differences in responses may reflect both biotic (i.e. initial community composition) and abiotic factors (currents and residence time of salinity gradients). Our results emphasize that desalination brine discharges that include chemicals such as iron-hydroxide and polyphosphonates can induce physiological and compositional changes in the microbial community. With the expansion of desalination facilities worldwide such shifts in composition and function of the microbial communities may destabilize and change local aquatic food webs and should thus be monitored.

Trichodesmium's strategies to alleviate phosphorus limitation in the future acidified oceans.

Global warming may exacerbate inorganic nutrient limitation, including phosphorus (P), in the surface waters of tropical oceans that are home to extensive blooms of the marine diazotrophic cyanobacterium, Trichodesmium. We examined the combined effects of P limitation and pCO(2), forecast under ocean acidification scenarios, on Trichodesmium erythraeum IMS101 cultures. We measured nitrogen acquisition,glutamine synthetase activity, C uptake rates, intracellular Adenosine Triphosphate (ATP) concentration and the pool sizes of related key proteins. Here, we present data supporting the idea that cellular energy re-allocation enables the higher growth and N(2) fixation rates detected in Trichodesmium cultured under high pCO(2). This is reflected in altered protein abundance and metabolic pools. Also modified are particulate organic carbon and nitrogen production rates,enzymatic activities, and cellular ATP concentrations. We suggest that adjusting these cellular pathways to changing environmental conditions enables Trichodesmium to compensate for low P availability and to thrive in acidified oceans. Moreover, elevated pCO(2) could provide Trichodesmium with a competitive dominance that would extend its niche, particularly in P-limited regions of the tropical and subtropical oceans.

Iron limitation in the marine cyanobacterium Trichodesmium reveals new insights into regulation of photosynthesis and nitrogen fixation.

* As iron (Fe) deficiency is a main limiting factor of ocean productivity, its effects were investigated on interactions between photosynthesis and nitrogen fixation in the marine nonheterocystous diazotrophic cyanobacterium Trichodesmium IMS101. * Biophysical methods such as fluorescence kinetic microscopy, fast repetition rate (FRR) fluorimetry, and in vivo and in vitro spectroscopy of pigment composition were used, and nitrogenase activity and the abundance of key proteins were measured. * Fe limitation caused a fast down-regulation of nitrogenase activity and protein levels. By contrast, the abundance of Fe-requiring photosystem I (PSI) components remained constant. Total levels of phycobiliproteins remained unchanged according to single-cell in vivo spectra. However, the regular 16-kDa phycoerythrin band decreased and finally disappeared 16-20 d after initiation of Fe limitation, concomitant with the accumulation of a 20-kDa protein cross-reacting with the phycoerythrin antibody. Concurrently, nitrogenase expression and activity increased. Fe limitation dampened the daily cycle of photosystem II (PSII) activity characteristic of diazotrophic Trichodesmium cells. Further, it increased the number and prolonged the time period of occurrence of cells with elevated basic fluorescence (F(0)). Additionally, it increased the effective cross-section of PSII, probably as a result of enhanced coupling of phycobilisomes to PSII, and led to up-regulation of the Fe stress protein IsiA. * Trichodesmium survives short-term Fe limitation by selectively down-regulating nitrogen fixation while maintaining but re-arranging the photosynthetic apparatus.

Synchronization of cell death in a dinoflagellate population is mediated by an excreted thiol protease.

Regulated programmed cell death (PCD) processes have been documented in several phytoplankton species and are hypothesized to play a role in population dynamics. However, the mechanisms leading to the coordinated collapse of phytoplankton blooms are poorly understood. We showed that the collapse of the annual bloom of Peridinium gatunense, an abundant dinoflagellate in Lake Kinneret, Israel, is initiated by CO2 limitation followed by oxidative stress that triggers a PCD-like cascade. We provide evidences that a protease excreted by senescing P. gatunense cells sensitizes younger cells to oxidative stress and may consequently trigger synchronized cell death of the population. Ageing of the P. gatunense cultures was characterized by a remarkable rise in DNA fragmentation and enhanced sensitivity to H2O2. Exposure of logarithmic phase (young) cultures to conditioning media from stationary phase (old) cells sensitized them to H2O2 and led to premature massive cell death. We detected the induction of specific extracellular protease activity, leupeptin-sensitive, in ageing cultures and in lake waters during the succession of the P. gatunense bloom. Partial purification of the conditioned media revealed that this protease activity is responsible for the higher susceptibility of young cells to oxidative stress. Inhibition of the protease activity lowered the sensitivity to oxidative stress, whereas application of papain to logarithmic phase P. gatunense cultures mimicked the effect of the spent media and enhanced cell death. We propose a novel mechanistic framework by which a population of unicellular phytoplankton orchestrates a coordinated response to stress, thereby determine the fate of its individuals.