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.     

The central role of selenium in the biochemistry and ecology of the harmful pelagophyte,Aureococcus anophagefferens

The trace element selenium (Se) is required for the biosynthesis of selenocysteine (Sec), the 21st amino acid in the genetic code, but its role in the ecology of harmful algal blooms (HABs) is unknown. Here, we examined the role of Se in the biology and ecology of the harmful pelagophyte, Aureococcus anophagefferens, through cell culture, genomic analyses, and ecosystem studies. This organism has the largest and the most diverse selenoproteome identified to date that consists of at least 59 selenoproteins, including known eukaryotic selenoproteins, selenoproteins previously only detected in bacteria, and novel selenoproteins. The A. anophagefferens selenoproteome was dominated by the thioredoxin fold proteins and oxidoreductase functions were assigned to the majority of detected selenoproteins. Insertion of Sec in these proteins was supported by a unique Sec insertion sequence. Se was required for the growth of A. anophagefferens as cultures grew maximally at nanomolar Se concentrations. In a coastal ecosystem, dissolved Se concentrations were elevated before and after A. anophagefferens blooms, but were reduced by >95% during the peak of blooms to 0.05 nℳ. Consistent with this pattern, enrichment of seawater with selenite before and after a bloom did not affect the growth of A. anophagefferens, but enrichment during the peak of the bloom significantly increased population growth rates. These findings demonstrate that Se inventories, which can be anthropogenically enriched, can support proliferation of HABs, such as A. anophagefferens through its synthesis of a large arsenal of Se-dependent oxidoreductases that fine-tune cellular redox homeostasis.     

Dynamics of the ‘echo’ effect in a phytoplankton system with nitrogen fixation

Consideration of nitrogen fixation adds a positive nonlinear feedback to plankton ecosystem models. We investigate the consequences of this feedback for secondary phytoplankton blooms and the response of phytoplankton dynamics to physical forcing. The dynamics of phytoplankton, Trichodesmium (the nitrogen fixer), and nutrients is modeled with a system of three differential equations. The model includes two types of nonlinear interactions: the competition of phytoplankton and Trichodesmium for light, and the positive feedback resulting from Trichodesmium recycling. A typical simulation of the model in time, with forcing by a varying mixed-layer depth, reveals a clear successional sequence including a secondary or ‘echo’ bloom of the phytoplankton. We explain this sequence of events through the stability analysis of three different steady states of the model. Our analysis shows the existence of a critical biological parameter, the ratio of normalized growth rates, determining the occurrence of ‘echo’ blooms and the specific sequence of events following a physical perturbation. The interplay of positive and negative feedbacks appears essential to the timing and the type of events following such a perturbation.     

The effects of temperature increases on a temperate phytoplankton community — A mesocosm climate change scenario

Prior to the spring bloom in 2003 and 2004, batch temperature experiments of approximately 3 weeks' duration were carried out in land-based mesocosms in at the Espeland field station (Norway), with temperatures on average increased ~ 2.7-3 °C (T1) and ~ 5.2-5.6 °C (T2) above in situ fjord temperature (RM). The development in the chlorophyll concentrations showed an earlier bloom as a response to increased temperatures but the carbon biomass showed that the warmest treatment yielded the lowest biomass. This study indicates that a part of the relationship between temperature and spring bloom timing stems from a temperature-induced change in phytoplankton algal physiology (the efficiency of photosystem II, F_v/F_m, and growth rates, µ_max), i.e. a direct temperature effect. Data analysis performed on microscope identified and quantified species did not show a significant temperature influence on phytoplankton community composition. However, the HPLC data indicated that temperature changes of as little as 3 °C influence the community composition. In particular, these data showed that peridinin-containing dinoflagellates only increased in abundance in the heated mesocosms and that a prasinophycean bloom, which was undetected in the microscope analyses, occurred prior to the blooms of all other phytoplankton classes in all treatments. The microscope analyses did reveal a temperature effect on individual species distribution patterns. Thalassionema nitzschioides was more abundant in the warm treatments and, in the warmest treatment, the spring bloom forming Skeletonema marinoi comprised a smaller proportion of the diatom community than in the other treatments.     

Dynamics of bacterial community structure during blooms of Cochlodinium polykrikoides (Gymnodiniales,Dinophyceae) in Korean coastal waters

Recent studies of dinoflagellates have reported that blooms can be closely related to the characteristics of the associated bacteria, but studies of the correlation between the toxic dinoflagellate, Cochlodinium polykrikoides and their associated bacterial community composition has not been explored. To understand this correlation, changes in bacterial community structure through the evolution of a C. polykrikoides bloom in Korean coastal waters via clone library analysis were investigated. Although there were no apparent changes in physio-chemical factors during the onset of the C. polykrikoides bloom, the abundance of bacteria bourgeoned in parallel with C. polykrikoides densities. Alpha-, gamma-proteobacteria and Flavobacteria were found to be dominant phyletic groups during C. polykrikoides blooms. The proportion of gamma-proteobacteria was lower (11.8%) during peak of the bloom period compared to the post-bloom period (26.2%). In contrast, alpha-proteobacteria increased in dominance during blooms. Among the alpha-proteobacteria, members of Rhodobacterales abruptly increased from 38% of the alpha-proteobacteria before the bloom to 74% and 56% during the early bloom and peak bloom stages, respectively. Moreover, multiple sites concurrently hosting C. polykrikoides blooms also contained high portions of Rhodobacterales and principal component analysis (PCA) demonstrated that Rhodobacterales had a positive, significant correlation with C. polykrikoides abundances (p ≤ 0.01, Pearson correlation coefficients). Collectively, this study reveals the specific clades of bacteria that increase (Rhodobacterales) and decrease (gamma-proteobacteria) in abundance C. polykrikoides during blooms.     

Genetic diversity of Microcystis populations in a bloom and its relationship to the environmental factors in Qinhuai River,China

Cyanobacterial blooms, forming massive scum and various cyanotoxins, increasingly spread in a wide range of freshwater ecosystems. One heavy Microcystis bloom occurred in the entire Qinhuai River basin in 2010 summer for the first time. To determine the Microcystis populations and their spatial distributions along Qinhuai River, a molecular approach was applied by sequencing the DNA library based on the internal transcribed spacer sequences of 16-23S rRNA (ITS). The parsimony network (TCS) analysis showed that 9 groups were formed based on the main 24 genotypes, and each group was dominated by one highly represented root sequence. Marked changes in the composition and proportion of the Microcystis ITS genotype were detected from the upper to the lower reaches. The seed sources forming the bloom were probably located at 4 different locations. Furthermore, it was found that pH was the primary factor affecting the spatial distribution of the main genotype group among samples.     

Reduced glutathione export during programmed cell death of Neurospora crassa

In a previous study, we demonstrated that staurosporine (STS) induces programmed cell death (PCD) in the fungus Neurospora crassa and that glutathione has the capability of inhibiting both STS-induced reactive oxygen species (ROS) formation and cell death. Here, we further investigated the role of glutathione in STS-induced PCD in N. crassa and observed an efflux of reduced glutathione (GSH) together with a change in the cell internal redox state to a more oxidative environment. This event was also observed with another PCD inducer, phytosphingosine (PHS), although externally added GSH did not prevent PHS-induced PCD. The nature of ROS, detected under the experimental conditions at which GSH export occurred, is also different in the two systems, predominantly superoxide in the case of STS and hydrogen peroxide in the case of PHS. In both cases, GSH export preceded the alterations in the plasma membrane that lead to selective dye permeation. We conclude that glutathione export in the context of PCD is not exclusive of certain mammalian cells and can be extended to Fungi, being an early PCD event in N. crassa. In addition, STS and PHS induce different PCD pathways in this fungus and the role of GSH export in each of them is likely different.     

Summer diatom blooms in the North Pacific subtropical gyre: 2008-2009

The summertime North Pacific subtropical gyre has widespread phytoplankton blooms between Hawaii and the subtropical front (～30°N) that appear as chlorophyll (chl) increases in satellite ocean color data. Nitrogen-fixing diatom symbioses (diatom-diazotroph associations: DDAs) often increase 10(2)-10(3) fold in these blooms and contribute to elevated export flux. In 2008 and 2009, two cruises targeted satellite chlorophyll blooms to examine DDA species abundance, chlorophyll concentration, biogenic silica concentration, and hydrography. Generalized observations that DDA blooms occur when the mixed layer depth is < 70 m are supported, but there is no consistent relationship between mixed layer depth, bloom intensity, or composition; regional blooms between 22-34°N occur within a broader temperature range (21-26°C) than previously reported. In both years, the Hemiaulus-Richelia and Rhizosolenia-Richelia DDAs increased 10(2)-10(3) over background concentrations within satellite-defined bloom features. The two years share a common trend of Hemiaulus dominance of the DDAs and substantial increases in the >10 μm chl a fraction (～40-90+% of total chl a). Integrated diatom abundance varied 10-fold over <10 km. Biogenic silica concentration tracked diatom abundance, was dominated by the >10 μm size fraction, and increased up to 5-fold in the blooms. The two years differed in the magnitude of the surface chl a increase (2009>2008), the abundance of pennate diatoms within the bloom (2009>2008), and the substantially greater mixed layer depth in 2009. Only the 2009 bloom had sufficient chl a in the >10 μm fraction to produce the observed ocean color chl increase. Blooms had high spatial variability; ocean color images likely average over numerous small events over time and space scales that exceed the individual event scale. Summertime DDA export flux noted at the Hawaii time-series Sta. ALOHA is probably a generalized feature of the eastern N. Pacific north to the subtropical front.     

Controls on the initiation and development of blooms of the dinoflagellate Cochlodinium polykrikoides Margalef in lower Chesapeake Bay and its tributaries

Massive blooms of the dinoflagellate Cochlodinium polykrikoides occur annually in the Chesapeake Bay and its tributaries. The initiation of blooms and their physical transport has been documented and the location of bloom initiation was identified during the 2007 and 2008 blooms. In the present study we combined daily sampling of nutrient concentrations and phytoplankton abundance at a fixed station to determine physical and chemical controls on bloom formation and enhanced underway water quality monitoring (DATAFLOW) during periods when blooms are known to occur. While C. polykrikoides did not reach bloom concentrations until late June during 2009, vegetative cells were present at low concentrations in the Elizabeth River (4 cells ml⁻¹) as early as May 27. Subsequent samples collected from the Lafayette River documented the increase in C. polykrikoides abundance in the upper branches of the Lafayette River from mid-June to early July, when discolored waters were first observed. The 2009 C. polykrikoides bloom began in the Lafayette River when water temperatures were consistently above 25 °C and during a period of calm winds, neap tides, high positive tidal residuals, low nutrient concentrations, and a low dissolved inorganic nitrogen (DIN) to dissolved inorganic phosphorous (DIP) ratio. The pulsing of nutrients associated with intense but highly localized storm activity during the summer months when water temperatures are above 25 °C may play a role in the initiation of C. polykrikoides blooms. The upper Lafayette River appears to be an important area for initiation of algal blooms that then spread to other connected waterways.     

The influence of cyanobacteria blooms on the attenuation of nitrogen throughputs in a Baltic coastal lagoon

We combined a mass balance approach with measurements of air–water and sediment–water nitrogen (N) exchange to better understand the mechanisms attenuating N throughputs in a eutrophic coastal lagoon. We were particularly interested in how seasonal shifts in external versus internal N fluxes and the transition from diatom- to cyanobacteria- dominated phytoplankton communities influence N storage and loss to the atmosphere. We found that on an annual basis almost all of the N removed by the lagoon was due to sediment storage following the spring diatom bloom. This period was characterized by high riverine inputs of dissolved inorganic nitrogen, high rates of assimilatory conversion to particulate nitrogen (PN), and net accrual of N in sediments. By contrast, the larger summer bloom was associated with low sediment N storage, which we attribute in part to the presence of positively-buoyant cyanobacteria. Low settling rates during cyanobacteria blooms favored export of PN to the Baltic Sea over sediment accrual in the lagoon. In addition, summer dinitrogen (N₂) fixation by cyanobacteria largely offset annual N₂ losses via denitrification. These findings show that cyanobacteria blooms diminish N attenuation within the lagoon by altering the balance of N exchange with the atmosphere and by promoting export of particulate N over sediment burial.     

Nitrogen Fixed By Cyanobacteria Is Utilized By Deposit-Feeders

Benthic communities below the photic zone depend for food on allochthonous organic matter derived from seasonal phytoplankton blooms. In the Baltic Sea, the spring diatom bloom is considered the most important input of organic matter, whereas the contribution of the summer bloom dominated by diazotrophic cyanobacteria is less understood. The possible increase in cyanobacteria blooms as a consequence of eutrophication and climate change calls for evaluation of cyanobacteria effects on benthic community functioning and productivity. Here, we examine utilization of cyanobacterial nitrogen by deposit-feeding benthic macrofauna following a cyanobacteria bloom at three stations during two consecutive years and link these changes to isotopic niche and variations in body condition (assayed as C:N ratio) of the animals. Since nitrogen-fixing cyanobacteria have δ¹⁵N close to -2‰, we expected the δ¹⁵N in the deposit-feeders to decrease after the bloom if their assimilation of cyanobacteria-derived nitrogen was substantial. We also expected the settled cyanobacteria with their associated microheterotrophic community and relatively high nitrogen content to increase the isotopic niche area, trophic diversity and dietary divergence between individuals (estimated as the nearest neighbour distance) in the benthic fauna after the bloom. The three surface-feeding species (Monoporeia affinis, Macoma balthica and Marenzelleria arctia) showed significantly lower δ¹⁵N values after the bloom, while the sub-surface feeder Pontoporeia femorata did not. The effect of the bloom on isotopic niche varied greatly between stations; populations which increased niche area after the bloom had better body condition than populations with reduced niche, regardless of species. Thus, cyanobacterial nitrogen is efficiently integrated into the benthic food webs in the Baltic, with likely consequences for their functioning, secondary production, transfer efficiency, trophic interactions, and intra- and interspecific competition.     

Quorum sensing control of phosphorus acquisition in Trichodesmium consortia

Colonies of the cyanobacterium Trichodesmium are abundant in the oligotrophic ocean, and through their ability to fix both CO₂ and N₂, have pivotal roles in the cycling of carbon and nitrogen in these highly nutrient-depleted environments. Trichodesmium colonies host complex consortia of epibiotic heterotrophic bacteria, and yet, the regulation of nutrient acquisition by these epibionts is poorly understood. We present evidence that epibiotic bacteria in Trichodesmium consortia use quorum sensing (QS) to regulate the activity of alkaline phosphatases (APases), enzymes used by epibionts in the acquisition of phosphate from dissolved-organic phosphorus molecules. A class of QS molecules, acylated homoserine lactones (AHLs), were produced by cultivated epibionts, and adding these AHLs to wild Trichodesmium colonies collected at sea led to a consistent doubling of APase activity. By contrast, amendments of (S)-4,5-dihydroxy-2,3-pentanedione (DPD)—the precursor to the autoinducer-2 (AI-2) family of universal interspecies signaling molecules—led to the attenuation of APase activity. In addition, colonies collected at sea were found by high performance liquid chromatography/mass spectrometry to contain both AHLs and AI-2. Both types of molecules turned over rapidly, an observation we ascribe to quorum quenching. Our results reveal a complex chemical interplay among epibionts using AHLs and AI-2 to control access to phosphate in dissolved-organic phosphorus.     

Bacterial community dynamics during a bloom caused by Akashiwo sanguinea in the Xiamen sea area,China

Phytoplankton blooms are a worldwide ecological problem and one of the major algae that cause phytoplankton blooms is Akashiwo sanguinea. Though much research has addressed the abiotic causes (e.g. growth condition) of A. sanguinea blooms, few studies have examined the dynamics of microbial communities associated with these blooms. In this study, polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysis of 16S rDNA genes was used to document changes in the phylogenetic diversity of microbial communities associated with an A. sanguinea bloom that occurred in the Xiamen sea in May 2010. Surface sea water was sampled once a day within five consecutive days at four sites, and the microbial community composition was determined using DGGE. Sea water concentrations of chlorophyll a, nitrate and phosphate were also measured. The results indicated that the A. sanguinea bloom was probably stimulated by low salinity (26–30‰) and ended probably because inorganic nutrients were consumed and resulted in a N/P ratio unfavorable for this alga. Gammaproteobacteria populations increased significantly during bloom declines and then decreased post-bloom. Divergences in the microbial community composition during different bloom periods were the result of changes in Candidatus, Pelagibacter, Alteromonas, Rhodobacteraceae, Vibrio and Pseudoalteromonas populations. Sediminimonas qiaohouensis was the first bacterium shown to be significantly negatively correlated with A. sanguinea concentration. This study indicated that bacteria may play an important role in A. sanguinea–bloom regulation and provides a deeper insight into bacterial community succession during and after an A. sanguinea–bloom.     

Deficient plastidic fatty acid synthesis triggers cell death by modulating mitochondrial reactive oxygen species

Programmed cell death (PCD) is of fundamental importance to development and defense in animals and plants. In plants, a well-recognized form of PCD is hypersensitive response (HR) triggered by pathogens, which involves the generation of reactive oxygen species (ROS) and other signaling molecules. While the mitochondrion is a master regulator of PCD in animals, the chloroplast is known to regulate PCD in plants. Arabidopsis Mosaic Death 1 (MOD1), an enoyl-acyl carrier protein (ACP) reductase essential for fatty acid biosynthesis in chloroplasts, negatively regulates PCD in Arabidopsis. Here we report that PCD in mod1 results from accumulated ROS and can be suppressed by mutations in mitochondrial complex I components, and that the suppression is confirmed by pharmaceutical inhibition of the complex I-generated ROS. We further show that intact mitochondria are required for full HR and optimum disease resistance to the Pseudomonas syringae bacteria. These findings strongly indicate that the ROS generated in the electron transport chain in mitochondria plays a key role in triggering plant PCD and highlight an important role of the communication between chloroplast and mitochondrion in the control of PCD in plants.     

Impacts of a ‘black tide’ harmful algal bloom on rocky-shore intertidal communities on the West Coast of South Africa

Algal blooms commonly occur along the South African west coast. In March 1994 a dense bloom developed within St Helena Bay. Its subsequent decay caused near-shore hypoxia and elevated hydrogen sulphide levels, leading to it being termed a ‘black tide’. The bloom caused immediate massive intertidal mortalities (95% reduction of biomass), significant changes in community structure, and declines in richness and diversity. Only four taxa were not diminished: ephemeral algae, the gastropod Afrolittorina knysnaensis, the anemone Bunodactis reynaudi and the false limpet Siphonaria capensis. This limpet species subsequently increased at impacted sites relative to controls, probably reflecting reduced competition from other grazers. Ephemeral algae flourished during early recolonisation while grazers were depleted, but declined as grazers re-established. Manipulative experiments demonstrated that grazers do have this capability. Perennial algae also flourished, but lagged behind ephemerals, possibly contributing to their decline. Predators took longest to recover. Differences in the responses of species reflect differing tolerances, zonation patterns and biological interactions. Communities at control sites remained unchanged over the four-year study, and those exposed to a low intensity of the bloom were little affected, but communities exposed to its full intensity failed to recover fully within four years. Increasing frequency of harmful algal blooms along the west coast of South Africa could, therefore, significantly alter rocky-shore communities if intervals between bloom events become shorter than recovery times.     

The effect of ultrasound on the growth and viability of microalgae cells

Ultrasound has shown potential for both increasing microalgal lipid extraction yields and for the control of microalgal blooms through cell disruption. The effect of ultrasound on the viability of microalgae was investigated on the following species: Dunaliella salina, Chlamydomonas concordia and Nannochloropsis oculata. Sonication with a 20 kHz probe (0.086 W cm^?3) caused complete cell disruption of D. salina after 4 min. This microalgae species does not have a true cell wall. In the case of C. concordia which has a thin cell wall complete cell disruption under the same conditions took 16 min. Under the same conditions, there was no visible disruption of N. oculata, a species which has a thick cell wall. However spectro-fluorophotometer analysis of the sonicated suspension of N. oculata showed that although the cells were intact, the level of intracellular chlorophyll was reduced by ~10 %. This clearly indicated damage to the microalgal cell wall. After 16 min, treatment cultures of all three species remained viable. Programmed cell death (PCD) has been induced in some microalgal species to terminate algal blooms; ultrasonic application did not induce PCD in any species tested. The supernatant of sonicated D. salina and C. concordia has also been shown to be able to boost the growth of established cultures. These results provide important information concerning the uses of ultrasound in both the microalgal biofuels industry and for the control of microalgal blooms.     

Functional Specificity of CpG DNA-binding CXXC Domains in Mixed Lineage Leukemia

The MLL CXXC domain binds nonmethylated CpG-containing DNA and is essential for the oncogenic properties of MLL fusion proteins. To determine potential functional promiscuity of similar DNA binding domains, we replaced the MLL CXXC domain in the context of the leukemogenic MLL-AF9 fusion with CXXC domains from DNMT1, CGBP (CFP1), and MBD1, or with a methyl-CpG-binding domain (MBD) from MBD1. MLL(DNMT1 CXXC)-AF9 shows robust in vitro colony forming activity and in vivo leukemogenesis, similar to MLL-AF9. However, colony forming ability and leukemogenicity are abrogated in MLL-AF9 containing either the CGBP or MBD1 CXXC domains or the MBD1 MBD domain. Direct comparison of in vitro DNA binding affinity of the isolated CXXC or MBD domains demonstrated that MLL, DNMT1, and CGBP CXXC domains could each bind to unmethylated DNA but with differing affinity. In contrast, the isolated MBD1 CXXC and MBD1 MBD domains were unable to bind to the same DNA. However, all substituted domains still allowed targeting of the MLL fusions to the functionally important Hoxa9 locus in primary bone marrow progenitor cells. In addition to DNA binding activity, it was critical that specific CpG residues in the Hoxa9 locus were protected from methylation for leukemia development. This ultimately prevented histone 3 lysine 9 trimethylation (H3K9me3) of the locus and enabled Hoxa9 expression. These were properties shared by MLL and DNMT1 CXXC domains but not by CGBP CXXC or the other swapped fusions tested. We demonstrate that similar CXXC domains can be mechanistically distinguished by specificity of CpG nucleotides preferentially protected from DNA methylation.