Two malyngamides from the caribbean cyanobacterium Lyngbya majuscula

Chemical examination of two collections of the Caribbean cyanobacterium, Lyngbya majuscula, led us to isolate several antimicrobial agents. The most active was elemental sulphur, followed by the previously reported fatty acid (−)-7(S)-methoxytetradec-4(E)-enoate, and then by two new amides of this fatty acid, malyngamide D and malyngamide D acetate. The new compounds possess vinyl chloride and α,β-unsaturated enone functional groups. The structures of the new compounds are based on spectroscopic evidence including several 2D NMR techniques at high field as well as chemical interconversion.     

Response of the estuarine cyanobacterium <Emphasis Type="Italic">Lyngbya aestuarii</Emphasis> to UV-B radiation

Growth response and changes in the spectral properties of methanolic extract of an estuarine cyanobacterium, Lyngbya aestuarii Agardh, to UV-B radiation were studied. Increase in growth accompanied by increase in chlorophyll a, protein and carbohydrate content was observed up to 12 h of UV-B irradiation followed by a decline with further increase in the duration of UV exposure. Carotenoid content progressively increased with the UV-B dose. The organism synthesized, to a significant extent, mycosporine amino acid-like substances (MAAs) upon UV-B exposure. The cells in the trichome became coiled followed by formation of small bundles as a response to UV-B radiation. SDS protein profile of the UV irradiated cells showed repression of 20 and 22 kDa proteins. However, irradiation with UV-B for 6–24 h led to overproduction of 84, 73, 60, 46, 40, 37 KDa proteins, possibly conferring protection to the organism from UV-B. UV irradiated cells cultured in florescent light for up to 7 days showed revival from UV damage of the pigments and macromolecular contents, suggesting existence of a repair mechanism in the organism.     

Natural antifoulants from the marine cyanobacterium Lyngbya majuscula

Filamentous benthic marine cyanobacteria are a prolific source of structurally unique bioactive secondary metabolites. A total of 12 secondary metabolites, belonging to the mixed polyketide–polypeptide structural class, were isolated from the marine cyanobacterium, Lyngbya majuscula, and were tested to determine if they showed activity against barnacle larval settlement. The assays revealed four compounds, dolastatin 16 (1), hantupeptin C (4), majusculamide A (10), and isomalyngamide A (12), that showed moderate to potent anti-larval settlement activities, with EC₅₀ values ranging from 0.003 to 10.6 μg ml^?1. In addition, field testing conducted over a period of 28 days (using the modified Phytagel? method) based on the cyanobacterial compound, dolastatin 16, showed significantly reduced barnacle settlement as compared to controls at all the concentrations tested. The results of this study highlight the importance of marine cyanobacteria as an underexplored source of potential environmentally friendly antifoulants.     

Contemporaneous N2 Fixation and Oxygenic Photosynthesis in the Nonheterocystous Mat-Forming Cyanobacterium Lyngbya aestuarii

The nonheterocystous filamentous cyanobacterial genus Lyngbya is a widespread and frequently dominant component of marine microbial mats. It is suspected of contributing to relatively high rates of N₂ fixation associated with mats. The ability to contemporaneously conduct O₂-sensitive N₂ fixation and oxygenic photosynthesis was investigated in Lyngbya aestuarii isolates from a North Carolina intertidal mat. Short-term (<4-h) additions of the photosystem II (O₂ evolution) inhibitor 3(3,4-dichlorophenyl)-1,1-dimethylurea stimulated light-mediated N₂ fixation (nitrogenase activity), indicating potential inhibition of N₂ fixation by O₂ production. However, some degree of light-mediated N₂ fixation in the absence of 3(3,4-dichlorophenyl)-1,1-dimethylurea was observed. Electron microscopic immunocytochemical localization of nitrogenase, coupled to microautoradiographic studies of ¹⁴CO₂ fixation and cellular deposition of the tetrazolium salt 2,4,5-triphenyltetrazolium chloride, revealed that (i) nitrogenase was widely distributed throughout individual filaments during illuminated and dark periods, (ii) ¹⁴CO₂ fixation was most active in intercalary regions, and (iii) daylight 2,4,5-triphenyltetrazolium chloride reduction (formazan deposition) was most intense in terminal regions. Results suggest lateral partitioning of photosynthesis and N₂ fixation during illumination, with N₂ fixation being confined to terminal regions. During darkness, a larger share of the filament appears capable of N₂ fixation.     

Nitrogen fixation by the benthic freshwater cyanobacterium Lyngbya wollei

The ability of the benthic cyanobacterium Lyngbya wollei to fix nitrogen was studied using field samples and axenic cultures. L. wollei was collected and isolated from Lake Okeechobee, Florida, where it forms extensive mats. Rates of acetylene reduction up to 39.1 nmol mg dry wt⁻¹ h⁻¹ were observed for field samples. The maximum observed rate of acetylene reduction in axenic laboratory cultures was 200 nmol mg dry wt⁻¹ h⁻¹. Aerobic conditions limited nitrogen fixation activity, but dark/light cycles promoted the development of activity. Reduced oxygen levels appeared to be required for the development of significant levels of nitrogenase activity. The level of irradiance also had a significant impact on the level of activity. The potential significance of nitrogen fixation to Lyngbya production is discussed.     

Besarhanamides A and B from the marine cyanobacterium Lyngbya majuscula

Besarhanamides A (1) and B (2) are fatty acid amides purified from the marine cyanobacterium, Lyngbya majuscula, collected from Pulau Hantu, Singapore. The structure determination of these secondary metabolites was carried out using extensive 2D NMR spectral data as well as chemical manipulations including the Marfey's method. In addition, besarhanamide A exhibited moderate toxicity with LD(50) at 13 microM in the brine shrimp toxicity bioassay.     

Contemporaneous N(2) Fixation and Oxygenic Photosynthesis in the Nonheterocystous Mat-Forming Cyanobacterium Lyngbya aestuarii

The nonheterocystous filamentous cyanobacterial genus Lyngbya is a widespread and frequently dominant component of marine microbial mats. It is suspected of contributing to relatively high rates of N(2) fixation associated with mats. The ability to contemporaneously conduct O(2)-sensitive N(2) fixation and oxygenic photosynthesis was investigated in Lyngbya aestuarii isolates from a North Carolina intertidal mat. Short-term (<4-h) additions of the photosystem II (O(2) evolution) inhibitor 3(3,4-dichlorophenyl)-1,1-dimethylurea stimulated light-mediated N(2) fixation (nitrogenase activity), indicating potential inhibition of N(2) fixation by O(2) production. However, some degree of light-mediated N(2) fixation in the absence of 3(3,4-dichlorophenyl)-1,1-dimethylurea was observed. Electron microscopic immunocytochemical localization of nitrogenase, coupled to microautoradiographic studies of CO(2) fixation and cellular deposition of the tetrazolium salt 2,4,5-triphenyltetrazolium chloride, revealed that (i) nitrogenase was widely distributed throughout individual filaments during illuminated and dark periods, (ii) CO(2) fixation was most active in intercalary regions, and (iii) daylight 2,4,5-triphenyltetrazolium chloride reduction (formazan deposition) was most intense in terminal regions. Results suggest lateral partitioning of photosynthesis and N(2) fixation during illumination, with N(2) fixation being confined to terminal regions. During darkness, a larger share of the filament appears capable of N(2) fixation.     

Lagunamide C, a cytotoxic cyclodepsipeptide from the marine cyanobacterium Lyngbya majuscula

Lagunamide C (1) is a cytotoxic cyclodepsipeptide isolated from the marine cyanobacterium, Lyngbya majuscula, from the western lagoon of Pulau Hantu Besar, Singapore. The complete structural characterization of the molecule was achieved by extensive NMR spectroscopic analysis as well as chemical manipulations. Several methods, including the advanced Marfey’s method, a modified method based on derivatization with Mosher’s reagents and analysis using LC–MS, and the use of ³J_H–H coupling constant values, were utilized for the determination of its absolute configuration. Compound 1 is related to the aurilide-class of molecules and it differs mainly in the macrocyclic structure by having a 27 membered ring system due to additional methylene carbon in the polyketide moiety. Lagunamide C displayed potent cytotoxic activity against a panel of cancer cell lines, such as P388, A549, PC3, HCT8, and SK-OV3 cell lines, with IC₅₀ values ranging from 2.1 nM to 24.4 nM. Compound 1 also displayed significant antimalarial activity with IC₅₀ value of 0.29 μM when tested against Plasmodium falciparum. In addition, lagunamide C exhibited weak anti-swarming activity when tested at 100 ppm against the Gram-negative bacterial strain, Pseudomonas aeruginosa PA01.     

Hantupeptins B and C,cytotoxic cyclodepsipeptides from the marine cyanobacterium Lyngbya majuscula

Hantupeptins B (2) and C (3) were isolated, along with the previously reported hantupeptin A (1), from the marine cyanobacterium, Lyngbya majuscula, collected from Pulau Hantu Besar, Singapore. Their structures were elucidated by interpretation of extensive 1D and 2D NMR spectroscopic data. Compounds 2 and 3 are cyclic depsipeptides consisting of five α-amino/hydroxy acid residues, including phenyllactic acid, proline, N-methyl-valine, valine, N-methyl-isoleucine, and a β-hydroxy acid unit with different degrees of unsaturation at the terminal end of each molecule. The absolute configurations of the common amino acids and phenyllactic acid were determined by the advanced Marfey’s and chiral HPLC analyses, respectively. The complete stereochemistry of 3-hydroxy-2-methyl-7-octynoic acid moiety in hantupeptin A was elucidated by a combination of homonuclear J-resolved 2D NMR experiments and by Mosher’s method. Hantupeptins B and C showed moderate in vitro cytotoxicity when tested against MOLT-4 (leukemic) and MCF-7 (breast cancer) cell lines.     

Pitipeptolides C-F, antimycobacterial cyclodepsipeptides from the marine cyanobacterium Lyngbya majuscula from Guam

Pitipeptolides A (1) and B (2) are cyclic depsipeptides isolated from the marine cyanobacterium Lyngbya majuscula from Piti Bomb Holes, Guam. Additional analogues have now been isolated by revisiting larger collections of the same cyanobacterium. The four identified analogues, pitipeptolides C–F (3–6), are the tetrahydro analogue (3), an analogue with a lower degree of methylation (4) as well as two homologues (5 and 6) of pitipeptolide A. Their structures were elucidated using 2D NMR experiments, chiral HPLC analysis and comparison with pitipeptolide A. The identified analogues showed weaker cytotoxic activities compared to the two major parent compounds, pitipeptolides A (1) and B (2), against HT-29 colon adenocarcinoma and MCF7 breast cancer cells. On the other hand, pitipeptolide F (6) was the most potent pitipeptolide in a disc diffusion assay against Mycobacterium tuberculosis. The latter finding suggests that the structure of pitipeptolides could be optimized for selective antibacterial activity.     

Microbial diversity and diazotrophy associated with the freshwater non-heterocyst forming cyanobacterium Lyngbya robusta

Lake Atitlan, Guatemala, a freshwater lake in South America, experiences annually recurring blooms comprised of the planktic filamentous cyanobacterium Lyngbya robusta. Previous physiochemical characterisation of the bloom identified diurnal nitrogenase activity typical of non-heterocystous cyanobacteria, in addition to the low-level detection of the cyanotoxins cylindrospermopsin and saxitoxin. A molecular approach, combining deep sequencing of the 16S rRNA and nifH genes, was applied to a cyanobacteria-dominated sample collected during the extensive 2009 bloom. Lyngbya accounted for over 60 % of the total 16S rRNA sequences with the only other cyanobacterial species detected being the picophytoplankton Synechococcus. The remaining bacterial population was comprised of organisms typical of other eutrophic freshwater bodies, although the proportionate abundances were atypical. An obligate anaerobe Opitutus, not typically found in freshwater systems, was identified within the community which suggests it may have a role in enhancing nitrogen fixation. Primary nitrogen fixation was attributed to Lyngbya, with other putative nitrogen fixers, Desulfovibrio, Clostridium and Methylomonas, present at very low abundance.     

The limit of resistance to salinity in the freshwater cyanobacterium Microcystis aeruginosa is modulated by the rate of salinity increase

The overall mean levels of different environmental variables are changing rapidly in the present Anthropocene, in some cases creating lethal conditions for organisms. Under this new scenario, it is crucial to know whether the adaptive potential of organisms allows their survival under different rates of environmental change. Here, we used an eco‐evolutionary approach, based on a ratchet protocol, to investigate the effect of environmental change rate on the limit of resistance to salinity of three strains of the toxic cyanobacterium Microcystis aeruginosa. Specifically, we performed two ratchet experiments in order to simulate two scenarios of environmental change. In the first scenario, the salinity increase rate was slow (1.5‐fold increase), while in the second scenario, the rate was faster (threefold increase). Salinity concentrations ranging 7–10 gL^‐1 NaCl (depending on the strain) inhibited growth completely. However, when performing the ratchet experiment, an increase in salinity resistance (9.1–13.6 gL^‐1 NaCl) was observed in certain populations. The results showed that the limit of resistance to salinity that M. aeruginosa strains were able to reach depended on the strain and on the rate of environmental change. In particular, a higher number of populations were able to grow under their initial lethal salinity levels when the rate of salinity increment was slow. In future scenarios of increased salinity in natural freshwater bodies, this could have toxicological implications due to the production of microcystin by this species.     

The role of respiration during adaptation of the freshwater cyanobacterium Synechococcus 6311 to salinity

Growth of the freshwater cyanobacterium Synechococcus 6311 under saline conditions stimulated respiration tenfold during the first 24 h, while growth and photosynthesis were inhibited. The elevated respiration rate was seen under both light and dark conditions, was uncoupler and cyanide sensitive, and did not decrease upon salt removal. Membrane preparations from salt-grown cells exhibited a tenfold increase in cytochrome oxidase activity, while electron transfer rates from NADPH to cytochrome c only increased threefold. Cytochrome oxidase activities were correlated with levels of EPR detectable Cu2+ in the salt and control membranes. Sodium-driven proton (antiproter) gradients in salt-grown cells were sensitive to cyanide but not dicyclohexylcarbodiimide, indicating the direct role of respiratory electron transport in maintaining low intracellular sodium levels.     

Pilot scale culture of the marine cyanobacterium Lyngbya majuscula for its pharmaceutically-useful natural metabolite curacin A

A Caribbean collection of Lyngbya majuscula which produces the promising antimitotic agent, curacin A, was recollected in 1993 from Curaçao and adapted to laboratory culture. A variety of culture vessel configurations and conditions were examined for their effect on growth and production of curacin A. Using these partially optimized conditions, 29 strains of curacin A producing L. majuscula were evaluated for their production levels of curacin A employing an internal standard GC-MS analytical method. Five strains were more carefully characterized for their growth and curacin A production, and led to our selection of one reliable strain, ’19L‘, for further studies. Growth and curacin A production curves were established for this strain over a 30 day growth period. This directed a scale-up culture of 640 liters of L. majuscula strain 19L in four batches of 160 L each. All four batches grew well and produced significant quantities of curacin A. In tota1, 215.5 g wet weight of L. majuscula tissue were produced which gave an isolated yield of 132.5 mg of curacin A.     

Effects of low salinity on metamorphosis in estuarine colonial ascidians

Abstract. We studied the effects of brackish water on larval attachment, events of metamorphosis, and juvenile mortality in three colonial ascidian species that live in a Florida coastal lagoon. Eudistoma olivaceum and Eudistoma hepaticum are restricted in their adult distribution to areas of relatively high and constant salinity near inlets, whereas Ecteinascidia turbinata extends more than 20 km into the Indian River, where salinity can be much more variable. In all three species, metamorphosis proceeded more quickly at 33 ppt than at lower salinities. The thresholds for successful metamorphosis differed among species in a manner that corresponded to the adult distributions, with E. turbinata being capable of completing metamorphosis at salinities as low as 22 ppt, E. hepaticum as low as 24 ppt, and E. olivaceum as low as 26 ppt. Larvae of both Eudistoma species delayed settlement in very low salinity water, whereas those of E. turbinata settled very quickly, then failed to complete metamorphosis. Juvenile mortality at salinities lower than 22 ppt was 100% for all three species. Survival in salinities higher than 22 ppt was strongly correlated with salinity in E. olivaceum and E. hepaticum , but not E. turbinata.     

Factors influencing growth and toxin production by cultures of the freshwater cyanobacterium Lyngbya wollei Farlow ex Gomont

Collections of Lyngbya wollei were taken from Guntersville Reservoir, Alabama, over a period of three years. Healthy filaments were isolated and transferred to agar plates of Z-8 and LM6E media. Unialgal isolates were cultured for the study of growth and paralytic shellfish poison (PSP) production. Filaments were extracted and the toxins were detected using high performance liquid chromatography (HPLC) with post column oxidation followed by fluorescence detection. HPLC profiles show that laboratory cultures of L. wollei produced decarbamoyl gonyautoxin 2 and 3, plus several other PSP like toxins whose structures are under investigation. At 26 °C and a light intensity of 11 or 22 μmol m-2 s-1 optimum production of both biomass and toxins occurred. A decrease or increase in temperature or light flux caused a reduction in dry weight or toxicity. Compared to control levels, lower PO4-P and NO3-N and higher calcium levels gave rise to higher biomass and toxicity. Lower calcium, calcium- or PO4-P deficient medium and high NO3-N or PO4-P caused a large decrease in dry weight and toxicity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Differential response of NaCl-Resistant mutants of the cyanobacterium Nostoc muscorum to salinity and osmotic stresses

Summary In the parent strain of Nostoc muscorum, the percentage survival, nitrogenase activity and oxygenic photosynthesis were severely impaired by NaCl (ionic) and sucrose (non-ionic) stresses. Spontaneously occurring NaCl-Resistant mutant clones of the cyanobacterium N. muscorum were found to exhibit differential responses under ionic and non-ionic stresses. One of the mutants (NaCl-R) was found to show resistance in terms of percentage survival, nitrogenase activity and oxygenic photosynthesis under saline (ionic) as well as osmotic (non-ionic) stresses and showing compatible solute strategy for such adaptation. Another mutant (Na⁺-R) was found to show resistance only to salinity stress and showed an enhanced Na⁺-efflux system driven by H⁺. The Na⁺-R mutant differed from the NaCl-R mutant strain in the sense that it was sucrose sensitive.