Decomposition of four macrophytes in wetland sediments: Organic matter and nutrient decay and associated benthic processes

Decomposition rates of Phragmites australis, Carex riparia, Nuphar luteum and Salvinia natans and benthic processes were measured from December 2003 to December 2004 in a shallow wetland (Paludi di Ostiglia, Northern Italy) by means of litter bags and intact cores incubations. Decay rate was highest for N. luteum (k = 0.0152 d⁻¹), intermediate for S. natans (k = 0.0041 d⁻¹) and similar for P. australis (k = 0.0027 d⁻¹) and C. riparia (k = 0.0028 d⁻¹).Benthic metabolism followed a seasonal pattern with summer peaks of O₂ demand and TCO₂, CH₄ and NH₄⁺ efflux whilst soluble reactive phosphorus (SRP) fluxes were negligible also under hypoxic conditions, indicating that P was mainly retained by sediment. The initial C:P ratio was similar in N. luteum and S. natans (170) and significantly lower than that of P. australis and C. riparia (360). During the detritus decay P was progressively lost by N. luteum and S. natans tissues, whereas, after an initial leaching, it was probably re-used during the microbial decomposition of the more refractory P. australis and C. riparia detritus. Nuphar luteum, P. australis and S. natans had comparable initial C:N mass ratio (15), significantly lower than that of C. riparia (26). The C:N ratio was rather constant for N. luteum (12.9 ± 1.5) and S. natans (14.6 ± 0.9), decreased slightly to below 20 for C. riparia and increased up to 30 for P. australis. Overall, differences among species were likely due to the recalcitrance of decomposing detritus, whilst process rates were controlled by limitation of microbial processes by nutrients and electron acceptor availability.     

Molecular cloning of a multidomain cysteine protease and protease inhibitor precursor gene from the tobacco hornworm (Manduca sexta) and functional expression of the cathepsin F-like cysteine protease domain

A Manduca sexta (tobacco hornworm) cysteine protease inhibitor, MsCPI, purified from larval hemolymph has an apparent molecular mass of 11.5 kDa, whereas the size of the mRNA is very large (9 kilobases). MsCPI cDNA consists of a 9,273 nucleotides that encode a polypeptide of 2,676 amino acids, which includes nine tandemly repeated MsCPI domains, four cystatin-like domains and one procathepsin F-like domain. The procathepsin F-like domain protein was expressed in Escherichia coli and processed to its active mature form by incubation with pepsin. The mature enzyme hydrolyzed Z-Leu–Arg–MCA, Z-Phe–Arg–MCA and Boc–Val–Leu–Lys–MCA rapidly, whereas hydrolysis of Suc–Leu–Tyr–MCA and Z-Arg–Arg–MCA was very slow. The protease was strongly inhibited by MsCPI, egg-white cystatin and sunflower cystatin with K_i values in the nanomolar range. When the MsCPI tandem protein linked to two MsCPI domains was treated with proteases, it was degraded by the cathepsin F-like protease. However, tryptic digestion converted the MsCPI tandem protein to an active inhibitory form. These data support the hypothesis that the mature MsCPI protein is produced from the MsCPI precursor protein by trypsin-like proteases. The resulting mature MsCPI protein probably plays a role in the regulation of the activity of endogenous cysteine proteases.     

Heterologous expression in Escherichia coli of Neurospora crassa neutral trehalase as an active enzyme

Neutral trehalase from Neurospora crassa was expressed in Escherichia coli as a polypeptide of 84 kDa in agreement with the theoretical size calculated from the corresponding cDNA. The recombinant neutral trehalase, purified by affinity chromatography exhibited a specific activity of 80–150 mU/mg protein. Optima of pH and temperature were 7.0 and 30 °C, respectively. The enzyme was absolutely specific for trehalose, and was quite sensitive to incubation at 40 °C. The recombinant enzyme was totally dependent on calcium, and was inhibited by ATP, copper, silver, aluminium and cobalt. K_M was 42 mM, and V_max was 30.6 nmol of glucose/min. The recombinant protein was phosphorylated by cAMP-dependent protein kinase, but not significantly activated. Immunoblotting with polyclonal antiserum prepared against the recombinant protein showed that neutral trehalase protein levels increased during exponential phase of N. crassa growth and dropped at the stationary phase. This is the first report of a neutral trehalase produced in E. coli with similar biochemical properties described for fungi native neutral trehalases, including calcium-dependence.     

Bottom-up controls on a mixed-species HAB assemblage: A comparison of sympatric Chattonella subsalsa and Heterosigma akashiwo (Raphidophyceae) isolates from the Delaware Inland Bays, USA

Recent novel mixed blooms of several species of toxic raphidophytes have caused fish kills and raised health concerns in the highly eutrophic Inland Bays of Delaware, USA. The factors that control their growth and dominance are not clear, including how these multi-species HAB events can persist without competitive exclusion occurring. We compared and contrasted the relative environmental niches of sympatric Chattonella subsalsa and Heterosigma akashiwo isolates from the bays using classic Monod-type experiments. C. subsalsa grew over a temperature range from 10 to 30 °C and a salinity range of 5–30 psu, with optimal growth occurring from 20 to 30 °C and 15 to 25 psu. H. akashiwo had similar upper temperature and salinity tolerances but also lower limits, with growth occurring from 4 to 30 °C and 5 to 30 psu and optimal growth between 16 and 30 °C and 10 and 30 psu. These culture results were confirmed by field observations of bloom occurrences in the Inland Bays. Maximum nutrient-saturated growth rates (μ_max) for C. subsalsa were 0.6 d⁻¹ and half-saturation concentrations for growth (K_s) were 9 μM for nitrate, 1.5 μM for ammonium, and 0.8 μM for phosphate. μ_max of H. akashiwo (0.7 d⁻¹) was slightly higher than C. subsalsa, but K_s values were nearly an order of magnitude lower at 0.3 μM for nitrate, 0.3 μM for ammonium, and 0.2 μM for phosphate. H. akashiwo is able to grow on urea but C. subsalsa cannot, while both can use glutamic acid. Cell yield experiments at environmentally relevant levels suggested an apparent preference by C. subsalsa for ammonium as a nitrogen source, while H. akashiwo produced more biomass on nitrate. Light intensity affected both species similarly, with the same growth responses for each over a range from 100 to 600 μmol photons m⁻² s⁻¹. Factors not examined here may allow C. subsalsa to persist during multi-species blooms in the bays, despite being competitively inferior to H. akashiwo under most conditions of nutrient availability, temperature, and salinity.     

Growth phase-dependant enzyme profile of pyruvate catabolism and end-product formation in Clostridium thermocellum ATCC 27405

End-product synthesis and enzyme activities involved in pyruvate catabolism, H₂ synthesis, and ethanol production in mid-log (OD₆₀₀  0.25), early stationary (OD₆₀₀  0.5), and stationary phase (OD₆₀₀  0.7) cell extracts were determined in Clostridium thermocellum ATCC 27405 grown in batch cultures on cellobiose. Carbon dioxide, hydrogen, ethanol, acetate and formate were major end-products and their production paralleled growth and cellobiose consumption. Lactate dehydrogenase, pyruvate:formate lyase, pyruvate:ferredoxin oxidoreductase, methyl viologen-dependant hydrogenase, ferredoxin-dependant hydrogenase, NADH-dependant hydrogenase, NADPH-dependant hydrogenase, NADH-dependant acetaldehyde dehydrogenase, NADH-dependant alcohol dehydogenase, and NADPH-dependant alcohol dehydrogenase activities were detected in all extracts, while pyruate dehydrogenase and formate dehydrogenase activities were not detected. All hydrogenase activities decreased (2–12-fold) as growth progressed from early exponential to stationary phase. Alcohol dehydrogenase activities fluctuated only marginally (<45%), while lactate dehydrogenase, pyruvate:formate lyase, and pyruvate:ferredoxin oxidoreductase remained constant in all cell extracts. We have proposed a pathway involved in pyruvate catabolism and end-product formation based on enzyme activity profiles in conjunction with bioinformatics analysis.     

Structural elucidation of an exopolysaccharide from mycelial fermentation of a Tolypocladium sp. fungus isolated from wild Cordyceps sinensis

A novel polysaccharide designated EPS-1A with an average molecular weight around 40 kDa was fractionated and purified by anion-exchange and gel-filtration chromatography from the crude exopolysaccharide (EPS) isolated from fermentation broth of Cs-HK1, a Tolypocladium sp. fungus isolated from wild Cordyceps sinensis. The structural characteristics of EPS-1A were determined with various methods (e.g. GC, GC–MS, FT-IR, ¹H NMR and ¹³C NMR) and through acid hydrolysis, methylation, periodate-oxidation and Smith degradation. The results suggested that EPS-1A was composed of glucose, mannose and galactose at 15.2:3.6:1.0 M ratio. EPS-1A was a slightly branched polysaccharide and its backbone was composed of (1 → 6)-α-d-glucose residues (77%) and (1 → 6)-α-d-mannose residues (23%). Branching occurred at O-3 position of (1 → 6)-α-d-mannose residues of the backbone with (1 → 6)-α-d-mannose residues and (1 → 6)-α-d-glucose residues, and terminated with β-d-galactose residues.     

Investigation of subcellular acidic compartments using alpha-aminophosphonate 31P nuclear magnetic resonance probes

The ³¹P nuclear magnetic resonance (NMR) characteristics, toxicity, and cellular penetration of five linear or cyclic α-aminophosphonate highly sensitive pH probes were investigated in Dictyostelium discoideum cells and isolated rat hearts and were compared with three phosphonic acid derivatives. The line width broadening at pH pK_a, which was satisfactorily modelized for all compounds, was significantly limited in biological milieu for the new markers, affording a four- to sixfold better accuracy in pH determination. Cellular uptake or washout of nontoxic concentrations (<15 mM) of α-aminophosphonates occurred by rapid passive permeation, whereas standard probes required a much slower fluid-phase pinocytosis and transport processes that could ultimately lead to trapping. Using mild concentrations (<4 mM) three α-aminophosphonates having 6 < pK_a < 7 allowed an easy and simultaneous ³¹P NMR determination of cytosolic, acidic, and extracellular compartments in anoxic–reoxygenated or starving D. discoideum.     

Late Quaternary millennial-scale variability in pelagic aragonite preservation off Somalia

In order to better understand Late Quaternary pelagic aragonite preservation in the western Arabian Sea we have investigated a high-resolution sediment core 905 off Somalia. Pteropod preservation is enhanced in times of reduced monsoon-driven productivity, indicated by low amounts of C_org and low barium to aluminium (Ba/Al) ratios. All periods corresponding to Heinrich events in the North Atlantic are represented by maxima in shell preservation of the common pteropod Limacina inflata (LDX values < 2, except for H5-equivalent with a poorer shell preservation, LDX > 2.66). Good shell preservation is also found during stadials at 52.1–53.2, 36, 33.2, and 31.9 ka. Relative abundance of pteropods and their fragments in the coarse fraction reaches maxima during Marine Isotope Stage (MIS) 5.2, during time-equivalents of Heinrich events 4–6 and in stadials at  53,  42.5, and 41.4 ka.On longer time scales, the pteropod abundance corresponds to the ‘Indo-Pacific carbonate preservation type’ with poor preservation during interglacials and better preservation during glacials. Late MIS 5 to early MIS 4 sections (84.1–64.8 ka) and the Late Holocene interval (6.5–0 ka) of core 905 contain only traces of pteropods. The early Holocene (9.2–6.5 ka) part is characterized by low pteropod amounts. Between 64.8 and 43.4 ka strong fluctuations occur and an intermediate average relative pteropod abundance is revealed. Between 43.4 and 9.2 ka the highest amounts in relative pteropod abundance in core 905 are observed. Besides the regional monsoonal influence on deepwater chemistry, changes in deepwater circulation occurring on glacial/interglacial and stadial/interstadial time scales might have affected pteropod preservation. However, it remains elusive whether 1) deep water formation in the Arabian Sea, 2) inflow of Glacial North Atlantic Intermediate Water or 3) change in water mass properties of the Circumpolar Deep Water (which is the water mass currently bathing this site) contributed to the observed pteropod preservation pattern.     

Optimization of human d-amino acid oxidase expression in Escherichia coli

Human d-amino acid oxidase (hDAAO) is a flavoprotein that plays a key role in the pathophysiology of schizophrenia. So far, the biochemical characterization of this enzyme has been hampered by the difficulty of expressing it in a common heterologous host such as Escherichia coli. Increasing amounts of recombinant hDAAO are indeed required for the investigation of its structure–function relationships and for the screening of new inhibitors to be used in the treatment of schizophrenia. A recombinant hDAAO has been over-expressed in BL21(DE3)Star E. coli cells. By alternating screenings of medium components at flask level and investigating physiological parameters in 2 L controlled batch fermentations, an improved, robust and scalable microbial process was set up giving almost a 40- and 4-fold improvement in volumetric productivity and specific activity, respectively. Under these conditions 770 U/L culture hDAAO with a specific activity of 0.4 U/mg protein and a specific productivity of 24.9 U/g biomass were produced. Optimization of medium ingredients, of the time and the amount of inducer’s addition, pH control at the moment of induction and harvest, low mechanical shear stress regime during recombinant protein production, represent the factors concurring to achieve the reported expression level. Notably, this expression level is higher than any previously described production of hDAAOs. A yield of 100 mg of pure hDAAO/L culture thus became available in comparison to the 1–10 mg/L previously reported.     

Purification and characterization of hyperthermotolerant leucine aminopeptidase from Geobacillus thermoleovorans 47b

A thermophilic bacterium, which we designated as Geobacillus thermoleovorans 47b was isolated from a hot spring in Beppu, Oita Prefecture, Japan, on the basis of its ability to grow on bitter peptides as a sole carbon and nitrogen source. The cell-free extract from G. thermoleovorans 47b contained leucine aminopeptidase (LAP; EC 3.4.11.10), which was purified 164-fold to homogeneity in seven steps, using ammonium sulfate fractionation followed by the column chromatography using DEAE-Toyopearl, hydroxyapatite, MonoQ and Superdex 200 PC gel filtration, followed again by MonoQ and hydroxyapatite. The enzyme was a single polypeptide with a molecular mass of 42,977.2 Da, as determined by matrix-assisted laser desorption ionization and time-of-flight mass spectrometry, and was found to be thermostable at 90°C for up to 1 h. Its optimal pH and temperature were observed to be 7.6–7.8 and 60°C, respectively, and it had high activity towards the substrates Leu-p-nitroanilide (p-NA)(100%), Arg-p-NA (56.3%) and LeuGlyGly (486%). The K_m and V_max values for Leu-p-NA and LeuGlyGly were 0.658 mM and 25.0 mM and 236.2 mol min^–1 mg^–1 protein and 1,149 mol min^–1 mg^–1 protein, respectively. The turnover rate (k_cat) and catalytic efficiency (k_cat/ K_m) for Leu-p-NA and LeuGlyGly were 10,179 s^–1 and 49,543 s^–1 and 15,470 mM^–1 s^–1 and 1981.7 mM^–1 s^–1, respectively. The enzyme was strongly inhibited by EDTA, 1,10-phenanthroline, dithiothreitol, -mercaptoethanol, iodoacetate and bestatin; and its apoenzyme was found to be reactivated by Co²⁺ .     

Incision of trivalent chromium [Cr(III)]-induced DNA damage by Bacillus caldotenax UvrABC endonuclease

Some hexavalent chromium [Cr(VI)]-containing compounds are lung carcinogens. Once within cells, Cr(VI) is reduced to trivalent chromium [Cr(III)] which displays an affinity for both DNA bases and the phosphate backbone. A diverse array of genetic lesions is produced by Cr including Cr–DNA monoadducts, DNA interstrand crosslinks (ICLs), DNA–Cr–protein crosslinks (DPCs), abasic sites, DNA strand breaks and oxidized bases. Despite the large amount of information available on the genotoxicity of Cr, little is known regarding the molecular mechanisms involved in the removal of these lesions from damaged DNA. Recent work indicates that nucleotide excision repair (NER) is involved in the processing of Cr–DNA adducts in human and rodent cells. In order to better understand this process at the molecular level and begin to identify the Cr–DNA adducts processed by NER, the incision of CrCl₃ [Cr(III)]-damaged plasmid DNA was studied using a thermal-resistant UvrABC NER endonuclease from Bacillus caldotenax (Bca). Treatment of plasmid DNA with Cr(III) (as CrCl₃) increased DNA binding as a function of dose. For example, at a Cr(III) concentration of 1 μM we observed 2 Cr(III)–DNA adducts per plasmid. At this same concentration of Cr(III) we found that 17% of the plasmid DNA contained ICLs (0.2 ICLs/plasmid). When plasmid DNA treated with Cr(III) (1 μM) was incubated with Bca UvrABC we observed 0.8 incisions/plasmid. The formation of endonuclease IV-sensitive abasic lesions or Fpg-sensitive oxidized DNA bases was not detected suggesting that the incision of Cr(III)-damaged plasmid DNA by UvrABC was not related to the generation of oxidized DNA damage. Taken together, our data suggest that a sub-fraction of Cr(III)–DNA adducts is recognized and processed by the prokaryotic NER machinery and that ICLs are not necessarily the sole lesions generated by Cr(III) that are substrates for NER.     

Gas exchange of Ginkgo biloba leaves at different CO2 concentration levels

One and a half year-old Ginkgo saplings were grown for 2 years in 7 litre pots with medium fertile soil at ambient air CO₂ concentration and at 700 μmol mol⁻¹ CO₂ in temperature and humidity-controlled cabinets standing in the field. In the middle of the 2nd season of CO₂ enrichment, CO₂ exchange and transpiration in response to CO₂ concentration was measured with a mini-cuvette system. In addition, the same measurements were conducted in the crown of one 60-year-old tree in the field. Number of leaves/tree was enhanced by elevated CO₂ and specific leaf area decreased significantly.CO₂ compensation points were reached at 75–84 μmol mol⁻¹ CO₂. Gas exchange of Ginkgo saplings reacted more intensively upon CO₂ than those of the adult Ginkgo. On an average, stomatal conductance decreased by 30% as CO₂ concentration increased from 30 to 1000 μmol mol⁻¹ CO₂. Water use efficiency of net photosynthesis was positively correlated with CO₂ concentration levels. Saturation of net photosynthesis and lowest level of stomatal conductance was reached by the leaves of Ginkgo saplings at >1000 μmol mol⁻¹ CO₂. Acclimation of leaf net CO₂ assimilation to the elevated CO₂ concentration at growth occurred after 2 years of exposure. Maximum of net CO₂ assimilation was 56% higher at ambient air CO₂ concentration than at 700 μmol mol⁻¹ CO₂.     

Adult Brugia malayi 34 kDa (BMT-5) antigen offers Th1 mediated significant protection against infective larval challenge in Mastomys coucha

We earlier reported a sizeable protection conferred by ‘mitochondria rich’ (MT) fraction of adult B. malayi and the present study was planned to locate the candidate protective molecule/s in the active fraction. The MT fraction was subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and the antigen bands showing strong immune-reactivity with the resistant mastomys sera were assayed for their lymphoproliferative response using splenocytes of protected animals. Of the eight such protein bands, one sub fraction with a molecular weight of  34 kDa (BMT-5) produced utmost cellular proliferation and was therefore exploited for vaccination study. BMT-5 emulsified in Freund's adjuvant produced discernible protection causing 69 and 67% reductions in microfilaraemia and adult worm burden respectively along with sterilization of 68% of the recovered female parasites. Significant levels of filaria-specific and non-specific lymphoproliferation along with enhanced release of Th1 cytokines (TNF-α, IFN-γ and IL-2) by splenocytes were observed in the vaccinated mastomys in addition to elevated levels of antigen-specific IgG, IgG2a, IgG2b and IgA. The peritoneal macrophages of immunized animals also revealed enhanced nitric oxide production in the presence of BMT-5. The findings suggest that  34 kDa (BMT-5) molecules present in the MT fraction of adult B. malayi provided sizeable protection against infective larval challenge by generating a Th1 biased milieu in the host.     

Historical biogeography, phylogenetic relationships and intraspecific diversity of agamid lizards in the Central Asian deserts of Kazakhstan and Uzbekistan

The Central Asian agamid lizards are ecologically and morphologically diverse, occurring across a broad range of desert environments in this biogeographically important region. It is probable that past climatic shifts have significantly influenced the diversification patterns and distributions of the agamid lizards of this region. To assess this within a phylogenetic framework we sequenced a 1200 bp region of mitochondrial DNA and a 1200 bp nuclear gene (RAG-1), incorporating both inter- and intraspecific sampling across Central Asian agamids. Our topology and divergence time estimates support an Eocene origin of the Agaminae subfamily on the Indian subcontinent, coinciding with the collision of India into Eurasia. The onset of aridification in Central Asia during the Late Oligocene, resulting from the retreat of the Paratethys Sea and the intensified uplift of the Tibetan–Himalayan complex, probably played an important role in the diversification of Phrynocephalus, one of the three genera studied. Intensification of aridity and geologic events in the Plio-Pleistocene and Quaternary glacial cycling probably had a significant influence on intraspecific diversification patterns within Phrynocephalus.     

Isolation and characterization of a cysteine protease from the latex of Araujia hortorum fruits

A new protease (araujiain h l) was purified to mass spectroscopy homogeneity from the latex of Araujia hortorum Fourn. (Asclepiadaceae) fruits by ultracentrifugation and ion exchange chromatography. The enzyme has a molecular mass of 24,031 (mass spectrometry) and an isoelectric point higher than 9.3. The optimum pH range for casein hydrolysis was 8.0–9.5. The enzyme showed remarkable caseinolytic activity at high temperatures, although its thermal stability decayed rapidly. The proteinase was activated by thiol compounds and inhibited by common thiol-blocking reagents, particularly E-64 and HgCl₂, suggesting the enzyme belongs to the cysteine protease family. The concentration of active sites as determined by titration with E-64 was 3.3 M. When assayed on N--CBZ-amino acid-p-nitrophenyl esters, the enzyme showed higher preference for the glutamine derivative, followed by those of alanine, asparagine, glycine, and leucine, in decreasing order. Partial homology (36–48%) with other plant cysteine proteinases was observed in an internal fragment obtained by Protease V8 treatment.     

The Pandinus imperator haemolymph lipoprotein, an unusual phosphatidylserine carrying lipoprotein

The haemolymph lipoprotein of the scorpion, Pandinus imperator was isolated and characterised. Contrary to the lipoproteins of insects and the discoidal HDL-lipoproteins of a crayfish and polychaete, the Pandinus lipoprotein consists of three instead of two apoproteins (apoPiLp I = 230 kDa, apoPiLp II = 130 kDa and apoPiLp III = 120 kDa). The apolipoproteins are arranged in varying stoichiometries as judged by cross-linking experiments. In lipoprotein samples from individual animals, the two smaller subunits occurred in a 1:1 stoichiometry, while the relative amount of the 230 kDa peptide varied. The lipoprotein is a slightly heart-shaped HDL with a diameter of 15 nm. It is present in two densities of 1100 and 1190 kg/m³, of which the latter is by far more abundant. The native molecular mass was estimated to be 500 kDa. The lipid content was determined as 33.5% and consists of 70% neutral lipids and 30% phospholipids. Strikingly, 42.5% of the phospholipids is phosphatidylserine while phosphatidylcholine and phosphatidylethanolamine account for 55.1% and 2.3%, respectively. Carbohydrate analysis suggests the presence of only high-mannose-type N-glycans. N-glycan profiling shows glycans corresponding to a size of 8.0–11.5 hexose units.     

ICChI, a glycosylated chitinase from the latex of Ipomoea carnea

A multi-functional enzyme ICChI with chitinase/lysozyme/exochitinase activity from the latex of Ipomoea carnea subsp. fistulosa was purified to homogeneity using ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography. The enzyme is glycosylated (14–15%), has a molecular mass of 34.94 kDa (MALDI–TOF) and an isoelectric point of pH 5.3. The enzyme is stable in pH range 5.0–9.0, 80 °C and the optimal activity is observed at pH 6.0 and 60 °C. Using p-nitrophenyl-N-acetyl-β-d-glucosaminide, the kinetic parameters K_m, V_max, K_cat and specificity constant of the enzyme were calculated as 0.5 mM, 2.5 × 10⁻⁸ mol min⁻¹ μg enzyme⁻¹, 29.0 s⁻¹ and 58.0 mM⁻¹ s⁻¹ respectively. The extinction coefficient was estimated as 20.56 M⁻¹ cm⁻¹. The protein contains eight tryptophan, 20 tyrosine and six cysteine residues forming three disulfide bridges. The polyclonal antibodies raised and immunodiffusion suggests that the antigenic determinants of ICChI are unique. The first fifteen N-terminal residues G–E–I–A–I–Y–W–G–Q–N–G–G–E–G–S exhibited considerable similarity to other known chitinases. Owing to these unique properties the reported enzyme would find applications in agricultural, pharmaceutical, biomedical and biotechnological fields.     

Karlotoxin mediates grazing by Oxyrrhis marina on strains of Karlodinium veneficum

Karlodinium veneficum is a common member of temperate, coastal phytoplankton assemblages that occasionally forms blooms associated with fish kills. Here, we tested the hypothesis that the cytotoxic and ichthyotoxic compounds produced by K. veneficum, karlotoxins, can have anti-grazing properties against the heterotrophic dinoflagellate, Oxyrrhis marina. The sterol composition of O. marina (>80% cholesterol) renders it sensitive to karlotoxin, and does not vary substantially when fed different algal diets even for prey that are resistant to karlotoxin. At in situ bloom concentrations (10⁴–10⁵ K. veneficum ml⁻¹), grazing rates (cells ingested per Oxyrrhis h⁻¹) on toxic K. veneficum strain CCMP 2064 were 55% that observed on the non-toxic K. veneficum strain MD5. At lower prey concentrations typical of in situ non-bloom levels (<10³ cells ml⁻¹), grazing rates (cells ingested per Oxyrrhis h⁻¹) on toxic K. veneficum strain CCMP 2064 were 70–80% of rates on non-toxic strain MD5. Growth of O. marina was significantly suppressed when fed the toxic strain of K. veneficum. Experiments with mixed prey cultures, where non-toxic strain MD5 was fluorescently stained, showed that the presence of toxic strain CCMP 2064 inhibited grazing of O. marina on the co-occurring non-toxic strain MD5. Exogenous addition of a sub-lethal dose (100 ng ml⁻¹) of purified karlotoxin inhibited grazing of O. marina by approximately 50% on the non-toxic K. veneficum strain MD5 or the cryptophyte S. major. These results identify karlotoxin as an anti-grazing compound for those grazers with appropriate sterol composition (i.e., desmethyl sterols). This strategy is likely to be an important mechanism whereby growth of K. veneficum is uncoupled from losses due to grazing, allowing it to form ichthyotoxic blooms in situ.