Characterization of the fungicidal activity of Calothrix elenkinii using chemical methods and microscopy

An investigation was directed towards biochemical characterization of cyanobacterium Calothrix elenkinii and analysis of the chemical nature and mode of action of its fungicidal metabolite(s) against oomycete Pythium debaryanum. Biochemical characterization of the culture in terms of carbohydrate utilization revealed the facultative nature of C. elenkinii. Unique antibiotic markers were also found for this strain. 16S rDNA sequencing of the strain revealed 98% similarity with Calothrix sp. PCC7101. The fungicidal activity was tested by disc diffusion assay of different fractions of the culture filtrate. A minimum inhibitory concentration of 10 μl was recorded for ethyl acetate fraction of the 7-weeks old culture filtrates. HPLC, followed by NMR spectral analysis demonstrated the presence of a substituted benzoic acid in the ethyl acetate fraction. Microscopic examination revealed distinct granulation, followed by disintegration of the hyphae of Pythium sp., indicating the presence of an active metabolite in the culture filtrates of Calothrix sp. The fungicidal activity of C. elenkinii can be attributed to the presence of 3-acetyl-2-hydroxy-6-methoxy-4-methyl benzoic acid. This is the first report of a benzoic acid derivative having fungicidal activity in cyanobacteria.     

Molecular Characterization of a Fungicidal Endoglucanase from the Cyanobacterium Calothrix elenkinii

A gene responsible for fungicidal activity was identified in the cyanobacterial strain Calothrix elenkinii RPC1, which had shown promise as a biocontrol agent. Functional screening of the genomic library revealed fungicidal (against Pythium aphanidermatum) and endoglucanase activities in two clones. Sequencing revealed an open reading frame of 1,044 bp, encoding 348 amino acid residues with a predicted molecular weight of 38 kDa. Analysis of the deduced amino acid sequence of the putative gene (cael1) showed 99% similarity with the β-1,4-endoglucanase from Anabaena laxa RPAN8 and 97% with the glucanase belonging to the peptidase M20 family of Anabaena variabilis and Nostoc sp. PCC7120, respectively. The putative promoters, ribosomal binding sites and a signal peptide of 22 amino acid residues were identified, revealing the secretory nature of the protein. The phylogenetic tree indicated a close relationship of the gene with Bacillus sp. This study is the first to report on the characterization of an endoglucanase in Calothrix sp.     

Induction and Characterization of UV Resistant Calothrix braunii

A UV resistant mutant of Calothrix braunii has been isolated after repeated exposure to UV-C (254 nm) radiation. LD₅₀ for wild type against UV-B was 1.74 hand 100% lethality was achieved after 3.5 h. Whereas, UV resistant mutant showed LD₅₀ at 3.33 h and loss of complete survival after 5 h exposure. The growth rate of mutant was about 29 per cent greater than that of the wild type. The photosynthetic pigments, chlorophyll a and phycoerythrin were stimulated by 36.2 and 41.2 per cent, respectively over wild type. There were no differences in nitrogenase activity nitrate reductase activity, extracellular ammonia production and of plasmid DNA.     

Novel synthetic methods for N-cyano-1H-imidazole-4-carboxamides and their fungicidal activity

A novel synthetic method of N-cyanocarboxamides has been developed with advantages of mild reaction condition, simpler procedure and easy reactant-product isolation compared with the existing methods. Using this novel method, 16 new N-cyano-1H-imidazole-4-carboxamide derivatives were synthesized and their structures were characterized by spectrum analysis. Further antifungal activity study showed that most of the newly synthesized compounds have good antifungal activity selectively against Rhizoctonia solani among the six fungi tested. Particularly, compound 12h was identified as the most promising candidate with an EC(50) of 2.63 μg/mL against R. solani.     

Characterization and implications of the cell surface reactivity of Calothrix sp. strain KC97

The cell surface reactivity of the cyanobacterium Calothrix sp. strain KC97, an isolate from the Krisuvik hot spring, Iceland, was investigated in terms of its proton binding behavior and charge characteristics by using acid-base titrations, electrophoretic mobility analysis, and transmission electron microscopy. Analysis of titration data with the linear programming optimization method showed that intact filaments were dominated by surface proton binding sites inferred to be carboxyl groups (acid dissociation constants [pK(a)] between 5.0 and 6.2) and amine groups (mean pK(a) of 8.9). Sheath material isolated by using lysozyme and sodium dodecyl sulfate generated pK(a) spectra similarly dominated by carboxyls (pK(a) of 4.6 to 6.1) and amines (pK(a) of 8.1 to 9.2). In both intact filaments and isolated sheath material, the lower ligand concentrations at mid-pK(a) values were ascribed to phosphoryl groups. Whole filaments and isolated sheath material displayed total reactive-site densities of 80.3 x 10(-5) and 12.3 x 10(-5) mol/g (dry mass) of cyanobacteria, respectively, implying that much of the surface reactivity of this microorganism is located on the cell wall and not the sheath. This is corroborated by electrophoretic mobility measurements that showed that the sheath has a net neutral charge at mid-pHs. In contrast, unsheathed cells exhibited a stronger negative-charge characteristic. Additionally, transmission electron microscopy analysis of ultrathin sections stained with heavy metals further demonstrated that most of the reactive binding sites are located upon the cell wall. Thus, the cell surface reactivity of Calothrix sp. strain KC97 can be described as a dual layer composed of a highly reactive cell wall enclosed within a poorly reactive sheath.     

Phosphomonoesterase activity of the cyanobacterium (blue-green alga) Calothrix parietina

Cellular and extracellular phosphomonoesterase activities were compared in Calothrix parietina D550, a strain whose original environment has been studied in detail. Activity in both fractions became detectable at about the same stage in batch culture. Differences in the influence of environmental factors between the two were slight, suggesting a common origin. The optimum temperatures for cellular and extracellular activities were 40 degrees C and 30 degrees C, respectively, and the upper limits for detectable activity were 80 degrees C and 65 degrees C. The pH optimum for both cellular and extracellular activity was 10.0-10.2. When P-limited cultures were tested with p-nitrophenyl phosphate (pNPP) as substrate, Km values for cellular and extracellular activities were 43 and 33 microM pNPP, respectively. Eleven ions were tested for their influence on activity. In most cases the effect was low or negligible at concentrations likely to be present in nature or freshwater laboratory media. Where obvious effects occurred, these were usually apparent at lower concentrations with extracellular than cellular activity. One mM Ca led to a 40% increase in extracellular activity in comparison with 0.1 mM Ca, but had no effect on cellular activity. However, inorganic phosphate, which had a marked inhibitory effect at concentrations above 10 microM, brought about a similar response with cellular and extracellular activities (approximately 60% decrease with 100 microM).     

Bioengineered silver nanoparticles using Curvularia pallescens and its fungicidal activity against Cladosporium fulvum

Microorganisms based biosynthesis of nanomaterials has triggered significant attention, due to their great potential as vast source of the production of biocompatible nanoparticles (NPs). Such biosynthesized functional nanomaterials can be used for various biomedical applications. The present study investigates the green synthesis of silver nanoparticles (Ag NPs) using the fungus Curvularia pallescens (C. pallescens) which is isolated from cereals. The C. pallescens cell filtrate was used for the reduction of AgNO₃ to Ag NPs. To the best of our knowledge C. pallescens is utilized first time for the preparation of Ag NPs. Several alkaloids and proteins present in the phytopathogenic fungus C. pallescens were mainly responsible for the formation of highly crystalline Ag NPs. The as-synthesized Ag NPs were characterized by using UV–Visible spectroscopy, X-ray diffraction and transmission electron microscopy (TEM). The TEM micrographs have revealed that spherical shaped Ag NPs with polydisperse in size were obtained. These results have clearly suggested that the biomolecules secreted by C. pallescens are mainly responsible for the formation and stabilization of nanoparticles. Furthermore, the antifungal activity of the as-prepared Ag NPs was tested against Cladosporium fulvum, which is the major cause of a serious plant disease, known as tomato leaf mold. The synthesized Ag NPs displayed excellent fungicidal activity against the tested fungal pathogen. The extreme zone of reduction occurred at 50 μL, whereas, an increase in the reduction activity is observed with increasing the concentration of Ag NPs. These encouraging results can be further exploited by employing the as synthesized Ag NPs against various pathogenic fungi in order to ascertain their spectrum of fungicidal activity.     

Discrimination of DNA hybridization using chemical force microscopy.

Atomic force microscopy (AFM) can be used to probe the mechanics of molecular recognition between surfaces. In the application known as "chemical force" microscopy (CFM), a chemically modified AFM tip probes a surface through chemical recognition. When modified with a biological ligand or receptor, the AFM tip can discriminate between its biological binding partner and other molecules on a heterogeneous substrate. The strength of the interaction between the modified tip and the substrate is governed by the molecular affinity. We have used CFM to probe the interactions between short segments of single-strand DNA (oligonucleotides). First, a latex microparticle was modified with the sequence 3'-CAGTTCTACGATGGCAAGTC and epoxied to a standard AFM cantilever. This DNA-modified probe was then used to scan substrates containing the complementary sequence 5'-GTCAAGATGCTACCGTTCAG. These substrates consisted of micron-scale, patterned arrays of one or more distinct oligonucleotides. A strong friction interaction was measured between the modified tip and both elements of surface-bound DNA. Complementary oligonucleotides exhibited a stronger friction than the noncomplementary sequences within the patterned array. The friction force correlated with the measured strength of adhesion (rupture force) for the tip- and array-bound oligonucleotides. This result is consistent with the formation of a greater number of hydrogen bonds for the complementary sequence, suggesting that the friction arises from a sequence-specific interaction (hybridization) of the tip and surface DNA.     

Characterization and Implications of the Cell Surface Reactivity of Calothrix sp. Strain KC97

The cell surface reactivity of the cyanobacterium Calothrix sp. strain KC97, an isolate from the Krisuvik hot spring, Iceland, was investigated in terms of its proton binding behavior and charge characteristics by using acid-base titrations, electrophoretic mobility analysis, and transmission electron microscopy. Analysis of titration data with the linear programming optimization method showed that intact filaments were dominated by surface proton binding sites inferred to be carboxyl groups (acid dissociation constants [pK_a] between 5.0 and 6.2) and amine groups (mean pK_a of 8.9). Sheath material isolated by using lysozyme and sodium dodecyl sulfate generated pK_a spectra similarly dominated by carboxyls (pK_a of 4.6 to 6.1) and amines (pK_a of 8.1 to 9.2). In both intact filaments and isolated sheath material, the lower ligand concentrations at mid-pK_a values were ascribed to phosphoryl groups. Whole filaments and isolated sheath material displayed total reactive-site densities of 80.3 × 10⁻⁵ and 12.3 × 10⁻⁵ mol/g (dry mass) of cyanobacteria, respectively, implying that much of the surface reactivity of this microorganism is located on the cell wall and not the sheath. This is corroborated by electrophoretic mobility measurements that showed that the sheath has a net neutral charge at mid-pHs. In contrast, unsheathed cells exhibited a stronger negative-charge characteristic. Additionally, transmission electron microscopy analysis of ultrathin sections stained with heavy metals further demonstrated that most of the reactive binding sites are located upon the cell wall. Thus, the cell surface reactivity of Calothrix sp. strain KC97 can be described as a dual layer composed of a highly reactive cell wall enclosed within a poorly reactive sheath.     

Characterization of single-cell electroporation by using patch-clamp and fluorescence microscopy

Electroporation of single NG108-15 cells with carbon-fiber microelectrodes was characterized by patch-clamp recordings and fluorescence microscopy. To minimize adverse capacitive charging effects, the patch-clamp pipette was sealed on the cell at a 90(o) angle with respect to the microelectrodes where the applied potential reaches a minimum. From transmembrane current responses, we determined the electric field strengths necessary for ion-permeable pore formation and investigated the kinetics of pore opening and closing as well as pore open times. From both patch-clamp and fluorescence microscopy experiments, the threshold transmembrane potentials for dielectric breakdown of NG108-15 cells, using 1-ms rectangular waveform pulses, was approximately 250 mV. The electroporation pulse preceded pore formation, and analyte entry into the cells was dictated by concentration, and membrane resting potential driving forces. By stepwise moving a cell out of the focused field while measuring the transmembrane current response during a supramaximal pulse, we show that cells at a distance of approximately 30 microm from the focused field were not permeabilized.     

The prediction of the type of the biological activity of chemical compounds by using the methods of mathematical statistics

The shortcomings of available approaches to determination of the character of chemical compounds biological activity according to calculated, tabular and experimental parameters have been analyzed using statistic methods. Some ways of available algorithms improving are given.     

Structural traits of the antioxidant and fungicidal activity of steroid glycosides

To investigate the origin of antioxidant and antifungal properties of steroid glycosides (SG), a logico-structural analysis of the structure-activity relationships has been performed for 70 compounds using a set of computer programs STRAC. The following structural features responsible for antioxidant activity are revealed: 1) furostanol type of aglycon; 2) the presence of more than 4 branched monosaccharides in the carbohydrate chain; 3) the presence of glucose at C26 and OH-groups in the genin part of SG. Most characteristic for antifungal activity is the fragment genin-C3-Gal-Glc-Glc-Rha..., as well as the spirostanol type of aglycon. The computer analysis provided the basis for predicting a type of biological activity for 12 new compounds of the SG class.     

Synthesis and fungicidal activity of novel imidazole-based ketene dithioacetals

Novel imidazole-based ketene dithioacetals show impressive in planta activity against the economically important plant pathogens Alternaria solani, Botryotinia fuckeliana, Erysiphe necator and Zymoseptoria tritici. Especially derivatives of the topical antifungal lanoconazole, which bear an alkynyloxy or a heteroaryl group in the para-position of the phenyl ring, exhibit excellent control of the mentioned phytopathogens. These compounds inhibit 14α -demethylase in the sterol biosynthesis pathway of the fungi. Synthesis routes starting from either benzaldehydes or acetophenones as well as structure-activity relationships are discussed in detail.     

Peptides of the constant region of antibodies display fungicidal activity

Synthetic peptides with sequences identical to fragments of the constant region of different classes (IgG, IgM, IgA) of antibodies (Fc-peptides) exerted a fungicidal activity in vitro against pathogenic yeasts, such as Candida albicans, Candida glabrata, Cryptococcus neoformans, and Malassezia furfur, including caspofungin and triazole resistant strains. Alanine-substituted derivatives of fungicidal Fc-peptides, tested to evaluate the critical role of each residue, displayed unaltered, increased or decreased candidacidal activity in vitro. An Fc-peptide, included in all human IgGs, displayed a therapeutic effect against experimental mucosal and systemic candidiasis in mouse models. It is intriguing to hypothesize that some Fc-peptides may influence the antifungal immune response and constitute the basis for devising new antifungal agents.