Evaluation of Nostoc Strain ATCC 53789 as a Potential Source of Natural Pesticides

The cyanobacterium Nostoc strain ATCC 53789, a known cryptophycin producer, was tested for its potential as a source of natural pesticides. The antibacterial, antifungal, insecticidal, nematocidal, and cytotoxic activities of methanolic extracts of the cyanobacterium were evaluated. Among the target organisms, nine fungi (Armillaria sp., Fusarium oxysporum f. sp. melonis, Penicillium expansum, Phytophthora cambivora, P. cinnamomi, Rhizoctonia solani, Rosellinia, sp., Sclerotinia sclerotiorum, and Verticillium albo-atrum) were growth inhibited and one insect (Helicoverpa armigera) was killed by the extract, as well as the two model organisms for nematocidal (Caenorhabditis elegans) and cytotoxic (Artemia salina) activity. No antibacterial activity was detected. The antifungal activity against S. sclerotiorum was further studied with both extracts and biomass of the cyanobacterium in a system involving tomato as a host plant. Finally, the herbicidal activity of Nostoc strain ATCC 53789 was evaluated against a grass mixture. To fully exploit the potential of this cyanobacterium in agriculture as a source of pesticides, suitable application methods to overcome its toxicity toward plants and nontarget organisms must be developed.     

Antibacterial, antifungal and cytotoxic activity of terrestrial cyanobacterial strains from Serbia

Cyanobacteria are known to be a rich source of biologically active compounds some of which can have pharmaceutical importance. In this work we present the screening results of cyanobacterial strains for their antibacterial, antifungal, and cytotoxic activity. Cyanobacterial strains were isolated from various soil types in province of Vojvodina and Central Serbia, Republic of Serbia. The screening included 9 strains of Anabaena and 9 strains of Nostoc. Both, extracellular products (from the culture liquid) and cellular crude lipophilic extracts were tested against 13 bacterial strains and 8 fungal strains. Cytotoxic activity was tested against three human cell lines. Methanol extracts were prepared according to ?stensvik. Antibacterial and antifungal activities were determined measuring inhibition zone, 48 h after inoculation. The cytotoxic activity was determined by sulforhodamine B (SRB) colorimetric assay. Of all cyanobacterial strains tested, 52% showed some antifungal and 41% antibacterial activity. Two out of six tested strains possessed cytotoxic activity. The cytotoxic activity of Anabaena strain S12 was found both in culture liquid and crude cell extract. It occurred specifically between the 21st and 42nd day of cultivation against HeLa and MCF7 cells, but had no activity against cell line derived from a healthy tissue. A high percentage of the active strains among the tested strains justify the effort of screening cyanobacteria that are isolated from terrestrial environments. The most promising strains for the fur- ther study are Anabaena strain S12 which showed strong cytotoxic and antibacterial activity and Ana- baena strain S20 which produces a potent antifungal compound. The future work, besides further screening and chemical identification of the active compounds, should also include the development of culture techniques that would lead to more efficient production of biologically active compounds.     

Suboptimal growth temperatures enhance the biological activity of cultured cyanobacterium Gloeocapsa sp.

The cytotoxic, antibacterial, and antifungal activities of cyanobacterium Gloeocapsa sp. strain Gacheva 2007/R-06/1 were investigated and the possibility for an enhancement of these activities by changing the culture conditions evaluated. Fatty acids of this cyanobacterium were found to be active against Streptococcus pyogenes. Exopolysaccharides inhibited the growth of both Gram-positive and Gram-negative bacteria and the fungus Candida albicans. Both exopolysaccharides and fatty acid mixtures also significantly decreased the viability of human cervical carcinoma cells, HeLa. Greater biological activities were exhibited by Gloeocapsa sp., cultured at suboptimal temperatures (15–26°C) than at optimal and supraoptimal ones. In comparison with higher light intensity, the low-light cultivation stimulated the cytotoxicity of the fatty acids. In general, low temperatures decreased the growth of Gloeocapsa sp., but promoted its biological activity. Prolonged cultivation also had a beneficial impact on the bioactivity. Compared to 4 days, the 17-day cultivation resulted in fourfold higher antibacterial and antifungal activities of exopolysaccharides and more than twice increases in their cytotoxicity. The study revealed that this cyanobacterial isolate is a new source of natural products with potential for pharmacological and medical applications.     

The Phenolic Contents and Antimicrobial Activities of Some Marine Algae from the Mediterranean Sea (Algeria)

In this study, three marine algae collected from western coast of algerian mediteranean sea (Ulva lactuca, Dictyota dichotoma, and Corallina elongata) were tested using the agar-well diffusion method for their production of antibacterial and antifungal agents on various organisms that cause diseases of humans and plants (Eschirichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Salmonella sp, Candida albicans, and Penicillium sp.). The total phenol content and antimicrobial activity were determined using different crude seaweeds extracts (methanol, diethylether, and chloroform). The results show that the chloroform extracts of (Ulva lactuca and Corallina elongata) had the highest activity against E. coli and Salmonella sp. The methanol extract obtained from (Ulva lactuca, Dictyota dichotoma, and Corallina elongata) showed antifungal activity for Candida albicans. The results of the study revealed that the seaweeds from Algeria appear to have immense potential as a source of antibacterial and antifungal compounds; they can be used in treating diseases caused by these organisms.     

Hydrogenases in Nostoc sp. Strain PCC 73102, a Strain Lacking a Bidirectional Enzyme

The present study was carried out in order to examine and characterize the bidirectional hydrogenase in the cyanobacterium Nostoc sp. strain PCC 73102. Southern hybridizations with the probes Av1 and Av3 (hoxY and hoxH, bidirectional hydrogenase small and large subunits, respectively) revealed the occurrence of corresponding sequences in Anabaena variabilis (control), Anabaena sp. strain PCC 7120, and Nostoc muscorum but not in Nostoc sp. strain PCC 73102. As a control, hybridizations with the probe hup2 (hupL, uptake hydrogenase large subunit) demonstrated the presence of a corresponding gene in all the cyanobacteria tested, including Nostoc sp. strain PCC 73102. Moreover, with three different growth media, a bidirectional enzyme that was functional in vivo was observed in N. muscorum, Anabaena sp. strain PCC 7120, and A. variabilis, whereas Nostoc sp. strain PCC 73102 consistently lacked any detectable in vivo activity. Similar results were obtained when assaying for the presence of an enzyme that is functional in vitro. Native polyacrylamide gel electrophoresis followed by in situ hydrogenase activity staining was used to demonstrate the presence or absence of a functional enzyme. Again, bands corresponding to hydrogenase activity were observed for N. muscorum, Anabaena sp. strain PCC 7120, and A. variabilis but not for Nostoc sp. strain PCC 73102. In conclusion, we were unable to detect a bidirectional hydrogenase in Nostoc sp. strain PCC 73102 with specific physiological and molecular techniques. The same techniques clearly showed the presence of an inducible bidirectional enzyme and corresponding structural genes in N. muscorum, Anabaena sp. strain PCC 7120, and A. variabilis. Hence, Nostoc sp. strain PCC 73102 seems to be an unusual cyanobacterium and an interesting candidate for future biotechnological applications.     

Characterization of genes for an alternative nitrogenase in the cyanobacterium Anabaena variabilis.

Anabaena variabilis ATCC 29413 is a heterotrophic, nitrogen-fixing cyanobacterium that has been reported to fix nitrogen and reduce acetylene to ethane in the absence of molybdenum. DNA from this strain hybridized well at low stringency to the nitrogenase 2 (vnfDGK) genes of Azotobacter vinelandii. The hybridizing region was cloned from a lambda EMBL3 genomic library of A. variabilis, mapped, and sequenced. The deduced amino acid sequences of the vnfD and vnfK genes of A. variabilis showed only about 56% similarity to the nifDK genes of Anabaena sp. strain PCC 7120 but were 76 to 86% similar to the anfDK or vnfDK genes of A. vinelandii. The organization of the vnf gene cluster in A. variabilis was similar to that of A. vinelandii. However, in A. variabilis, the vnfG gene was fused to vnfD; hence, this gene is designated vnfDG. A vnfH gene was not contiguous with the vnfDG gene and has not yet been identified. A mutant strain, in which a neomycin resistance cassette was inserted into the vnf cluster, grew well in a medium lacking a source of fixed nitrogen in the presence of molybdenum but grew poorly when vanadium replaced molybdenum. In contrast, the parent strain grew equally well in media containing either molybdenum or vanadium. The vnf genes were transcribed in the absence of molybdenum, with or without vanadium. The vnf gene cluster did not hybridize to chromosomal DNA from Anabaena sp. strain PCC 7120 or from the heterotrophic strains, Nostoc sp. strain Mac and Nostoc sp. strain ATCC 29150. A hybridizing ClaI fragment very similar in size to the A. variabilis ClaI fragment was present in DNA isolated from several independent, cultured isolates of Anabaena sp. from the Azolla symbiosis.     

Photosynthetic CO2 fixation and ribulose bisphosphate carboxylase/oxygenase activity of Nostoc sp. strain UCD 7801 in symbiotic association with Anthoceros punctatus.

The cyanobacterium Nostoc sp. strain UCD 7801, immediately after separation from pure cultures of a reconstituted symbiotic association with the bryophyte Anthoceros punctatus, exhibited a rate of light-dependent CO2 fixation that was eightfold lower than that measured in the free-living growth state. Ribulose bisphosphate carboxylase/oxygenase (RuBPC/O) specific activity was also eightfold lower in cell extracts of symbiotic strain 7801 relative to that in free-living cultures. The in vitro activity from symbiotic strain 7801 could not be increased by incubation under the standard RuBPC/O activation conditions. Polyclonal antibodies against the RuBPC/O large subunit were used in an enzyme-linked immunosorbent assay to determine that RuBPC/O accounted for 4.3 and 5.2% of the total protein in cell extracts of strain 7801 grown in symbiotic and free-living states, respectively. The results imply that the regulation of RuBPC/O activity in the symbiotic growth state is by a posttranslational mechanism rather than by an alteration in RuBPC/O protein synthesis. The amount of carboxyarabinitol bisphosphate required to irreversibly inhibit RuBPC/O activity of sybiotic cell extracts was 80% of that required for extracts of free-living cultures. This result indicates that any covalent modification of RuBPC/O in symbiotically associated Nostoc cells did not interfere with the ribulose bisphosphate binding site, since inactive enzyme also bound carboxyarabinitol bisphosphate.     

Molecular evolution of PAS domain-containing proteins of filamentous cyanobacteria through domain shuffling and domain duplication.

When the entire genome of a filamentous heterocyst-forming N2-fixing cyanobacterium, Anabaena sp. PCC 7120 (Anabaena) was determined in 2001, a large number of PAS domains were detected in signal-transducing proteins. The draft genome sequence is also available for the cyanobacterium, Nostoc punctiforme strain ATCC 29133 (Nostoc), that is closely related to Anabaena. In this study, we extracted all PAS domains from the Nostoc genome sequence and analyzed them together with those of Anabaena. Clustering analysis of all the PAS domains gave many specific pairings, indicative of evolutionary conservations. Ortholog analysis of PAS-containing proteins showed composite multidomain architecture in some cases of conserved domains and domains of disagreement between the two species. Further inspection of the domains of disagreement allowed us to trace them back in evolution. Thus, multidomain proteins could have been generated by duplication or shuffling in these cyanobacteria. The conserved PAS domains in the orthologous proteins were analyzed by structural fitting to the known PAS domains. We detected several subclasses with unique sequence features, which will be the target of experimental analysis.     

Alterations in the antibacterial potential of Synechococcus spp. PCC7942 under the influence of UV-B radiations on skin pathogens

Marine organisms are seen as a source of novel drugs and the discovery of new pharmaceutical is increasingly in demand. Cyanobacteria are regarded as a potential target for this as antibacterial, antiviral, antifungal, algicide and cytotoxic activities have been reported in these organisms. They have been identified as a new and rich source of bioactive compounds belonging to diversified groups. Radiation in the UV-B range interferes with various metabolic reactions by generating free radicals and active oxygen species. These deleterious compounds are inactivated by antioxidants. Among them are the carotenoids and phycocyanin which protect against photodynamic action in different ways. Stress plays an important role in the production of bioactive metabolites from organisms. Synechococcus spp. PCC7942 was studied for antibacterial activity against various pathogenic bacteria resistant to a number of available antibiotics after being exposed to UV-B radiation. The antibacterial activity of Synechococcus spp. PCC7942 was studied on five potent skin pathogens. The highest antibacterial activity was seen the methanol extracts of 24 h UV-B exposed cultures of Synechococcus spp. PCC7942. It can be concluded that there was moderate antibacterial activity. Results showed stress, solvent and dose-dependent activity. This antibacterial activity might be due to the enhanced synthesis of carotenoids and phycocyanin under UV-B stress. The purpose of the present study was to relate the inhibitory effects of the cyanobacterial compounds specifically on skin pathogens with exposure to UV-B radiation as UV protecting compounds are already reported in these organisms.     

Physiological sources of reductant for nitrogen fixation activity in Nostoc sp. strain UCD 7801 in symbiotic association with Anthoceros punctatus.

Pure cultures of the symbiotic cyanobacterium-bryophyte association with Anthoceros punctatus were reconstituted by using Nostoc sp. strain UCD 7801 or its 3-(3,4-dichlorophenol)-1,1-dimethylurea (DCMU)-resistant mutant strain, UCD 218. The cultures were grown under high light intensity with CO2 as the sole carbon source and then incubated in the dark to deplete endogenous reductant pools before measurements of nitrogenase activities (acetylene reduction). High rates of light-dependent acetylene reduction were obtained both before starvation in the dark and after recovery from starvation, regardless of which of the two Nostoc strains was reconstituted in the association. Rates of acetylene reduction by symbiotic tissue with the wild-type Nostoc strain decreased 99 and 96% after 28 h of incubation in the dark and after reexposure to light in the presence of 5 microM DCMU, respectively. Supplementation of the medium with glucose restored nitrogenase activity in the dark to a rate that was 64% of the illuminated rate. In the light and in the presence of 5 microM DCMU, acetylene reduction could be restored to 91% of the uninhibited rate by the exogenous presence of various carbohydrates. The rate of acetylene reduction in the presence of DCMU was 34% of the uninhibited rate of tissue in association with the DCMU-resistant strain UCD 218. This result implies that photosynthates produced immediately by the cyanobacterium can supply at least one-third of the reductant required for nitrogenase activity on a short-term basis in the symbiotic association. However, high steady-state rates of nitrogenase activity by symbiotic Nostoc strains appear to depend on endogenous carbohydrate reserves, which are presumably supplied as photosynthate from both A. punctatus tissue and the Nostoc strain.     

The solution structure of photosystem I accessory protein E from the cyanobacterium Nostoc sp. strain PCC 8009.

PsaE is a small basic subunit located on the stromal (cytoplasmic) side of photosystem I. In cyanobacteria, this subunit participates in cyclic electron transport and modulates the interactions of the complex with soluble ferredoxin. The PsaE protein isolated from the cyanobacterium Synechococcus sp. strain PCC 7002 adopts the beta topology of an SH3 domain, with five beta strands (betaA through betaE) and a turn of 3(10) helix between strands betaD and betaE [Falzone, C. J., Kao, Y.-H., Zhao, J., Bryant, D. A., and Lecomte, J. T. J. (1994) Biochemistry 33, 6052-6062]. The primary structure of the PsaE protein is strongly conserved across all oxygen-evolving photosynthetic organisms. However, variability in loop lengths, as well as N- or C-terminal extensions, suggests that the structure of a second representative PsaE subunit would be useful to characterize the interactions among photosystem I polypeptides. In this work, the solution structure of PsaE from the cyanobacterium Nostoc sp. strain PCC 8009 was determined by NMR methods. Compared to PsaE from Synechococcus sp. strain PCC 7002, this PsaE has a seven-residue deletion in the loop connecting strands betaC and betaD, and an eight-residue C-terminal extension. Angular and distance restraints derived from homonuclear and heteronuclear NMR experiments were used to calculate structures by a distance-geometry/simulated-annealing protocol. A family of 20 structures (rmsd of 0.24 A in the regular secondary structure) is presented. Differences between the two cyanobacterial proteins are mostly confined to the CD loop region; the C-terminal extension is disordered. The thermodynamic stability of Nostoc sp. strain PCC 8009 PsaE toward urea denaturation was measured by circular dichroism and fluorescence spectroscopy, and thermal denaturation was monitored by UV absorption spectroscopy. Chemical and thermal denaturation curves are modeled satisfactorily with two-state processes. The DeltaG degrees of unfolding at room temperature is 12.4 +/- 0.3 kJ mol(-1) (pH 5), and the thermal transition midpoint is 59 +/- 1 degrees C (pH 7). Interactions with other proteins in the photosystem I complex may aid in maintaining PsaE in its native state under physiological conditions.     

Carbon-phosphorus bond cleavage activity in cell-free extracts of Enterobacter aerogenes ATCC 15038 and Pseudomonas sp. 4ASW.

Carbon-phosphorus bond cleavage activity was investigated in cell-free extracts of Enterobacter aerogenes ATCC 15038 (IFO 12010) and Pseudomonas sp. 4ASW, strains known to utilize a range of phosphonates as sole phosphorus source. In vitro phosphonatase activity was detected in extracts of both organisms; however extensive analysis failed to detect any organic product from phosphonates other than phosphonoacetal dehyde. Non-specific liberation of phosphate was observed in Pseudomonas sp. 4ASW, associated with a single fraction of FPLC-purified extract, and is believed to result from the activity of cellular phosphatases.     

Diversity and antimicrobial activities of the fungal endophyte community associated with the traditional Brazilian medicinal plant Solanum cernuum Vell. (Solanaceae)

The diversity and antimicrobial activity of endophytic fungi associated with the Brazilian medicinal plant Solanum cernuum Vell. were studied during summer and winter seasons. A total of 246 fungal isolates were obtained, including 225 filamentous fungi and 21 yeasts. They were identified by morphological, physiological, and molecular methods. Fifty-five different taxa represented by the phyla Ascomycota (33 taxa), Basidiomycota (21 taxa), and Zygomycota (one taxon) were identified. The most abundant taxa were closely related to Arthrobotrys foliicola , Colletotrichum gloeosporioides , Coprinellus radians , Glomerella acutata , Diatrypella frostii , Phoma glomerata , Mucor sp., Phlebia subserialis , Phoma moricola , Phanerochaete sordida , and Colletotrichum sp. A total of 265 fungal extracts were screened and 64 (26.01%) displayed antimicrobial activities. Among these extracts, 18 (28.12%) presented antibacterial and antifungal activities, 42 (65.62%) displayed selective antibacterial activity, and four (6.25%) exhibited only antifungal activity. The best values of minimum inhibitory concentration were obtained from extracts of Cryptococcus rajasthanensis , Glomerella acutata, Leptosphaeria sp., and Phoma glomerata ranging from 7.8 to 15.62?μg/mL. This study is the first survey of the endophytic fungi community associated with S. cernuum, and our results show that they can represent a promising source of bioactive compounds.     

Detection and characterization of cyanobacterial nifH genes.

The DNA sequence of a 359-bp fragment of nifH was determined for the heterocystous strains Anabaena sp. strain CA (ATCC 33047), Nostoc muscorum UTEX 1933, a Nostoc sp., Gloeothece sp. strain ATCC 27152, Lyngbya lagerheimii UTEX 1930, and Plectonema boryanum IU 594. Results confirmed that the DNA sequence of the 359-bp segment is sufficiently variable to distinguish cyanobacterial nifH genes from other eubacterial and arachaeobacterial nifH genes, as well as to distinguish heterocystous from nonheterocystous nifH genes. Nonheterocystous cyanobacterial nifH sequences were greater than 70 and 82% identical on the DNA and amino acid levels, respectively, whereas corresponding values for heterocystous cyanobacterial nifH sequences were 84 and 91%. The amplified nifH fragments can be used as DNA probes to differentiate between species, although there was substantial cross-reactivity between the nifH amplification products of some strains. However, an oligonucleotide designed from a sequence conserved within the heterocystous cyanobacteria hybridized primarily with the amplification product from heterocystous strains. The use of oligonucleotides designed from amplified nifH sequences shows great promise for characterizing assemblages of diazotrophs.     

Analysis of the hli gene family in marine and freshwater cyanobacteria

Certain cyanobacteria thrive in natural habitats in which light intensities can reach 2000 micromol photon m(-2) s(-1) and nutrient levels are extremely low. Recently, a family of genes designated hli was demonstrated to be important for survival of cyanobacteria during exposure to high light. In this study we have identified members of the hli gene family in seven cyanobacterial genomes, including those of a marine cyanobacterium adapted to high-light growth in surface waters of the open ocean (Prochlorococcus sp. strain Med4), three marine cyanobacteria adapted to growth in moderate- or low-light (Prochlorococcus sp. strain MIT9313, Prochlorococcus marinus SS120, and Synechococcus WH8102), and three freshwater strains (the unicellular Synechocystis sp. strain PCC6803 and the filamentous species Nostoc punctiforme strain ATCC29133 and Anabaena sp. [Nostoc] strain PCC7120). The high-light-adapted Prochlorococcus Med4 has the smallest genome (1.7 Mb), yet it has more than twice as many hli genes as any of the other six cyanobacterial species, some of which appear to have arisen from recent duplication events. Based on cluster analysis, some groups of hli genes appear to be specific to either marine or freshwater cyanobacteria. This information is discussed with respect to the role of hli genes in the acclimation of cyanobacteria to high light, and the possible relationships among members of this diverse gene family.     

Screening of nonpolyenic antifungal metabolites produced by clinical isolates of actinomycetes

The purpose of this work was to screen clinical isolates of actinomycetes producing nonpolyenic antifungals. This choice was made to limit the problem of rediscovery of well-known antifungal families, especially polyenic antifungals. One hundred and ten strains were tested, using two diffusion methods and two test media, against three yeast species and three filamentous fungi. Among 54 strains (49%) showing antifungal activity, five strains belonging to the genus Streptomyces were active against all test organisms and appeared promising. These results indicate that clinical and environmental isolates of actinomycetes could be an interesting source of antifungal bioactive substances. The production of nonpolyenic antifungal substances by these five active isolates was investigated using several criteria: antibacterial activity, ergosterol inhibition, and UV-visible spectra of active extracts. One active strain responded to all three selection criteria and produced potentially nonpolyenic antifungal metabolites. This strain was retained for further investigation, in particular, purification, structure elucidation, and mechanism of action of the active product.     

Sponge-associated sp. RM66 metabolome induction with N-acetylglucosamine: Antibacterial,antifungal and anti-trypanosomal activities

The marine sponge Amphimedon sp., collected from Hurghada (Egypt) was investigated for its sponge-derived actinomycetes diversity. Nineteen actinomycetes were cultivated and phylogenetically identified using 16S rDNA gene sequencing were carried out. The strains belong to genera Kocuria, Dietzia, Micrococcus, Microbacterium and Streptomyces. Many silent biosynthetic genes clusters were investigated using genome sequencing of actinomycete strains and has revealed in particular the genus Streptomyces that has indicated their exceptional capacity for the secondary metabolites production that not observed under classical cultivation conditions. In this study, the effect of N-acetylglucosamine on the metabolome of Streptomyces sp. RM66 was investigated using three actinomycetes media (ISP2, M1 and MA). In total, twelve extracts were produced using solid and liquid fermentation approaches. Liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) data were analysed using metabolomics tools to compare natural product production across all crude extracts. Our study highlighted the elicitation effect of N-acetylglucosamine on the secondary metabolite profiles of Streptomyces sp. RM66. These results highlight the of N-acetylglucosamine application as an elicitor to induce the cryptic metabolites and for increasing the chemical diversity. All the twelve extracts were tested for their antibacterial activity was tested against Staphylococcus aureus NCTC 8325, antifungal activity against Candida albicans 5314 (ATCC 90028) and anti-trypanosomal activity against Trypanosoma brucei brucei. Extract St1 showed the most potent one with activities 2.3, 3.2 and 4.7 ug/ml as antibacterial, antifungal and anti-trypanosomal, respectively.     

Discovery of rare and highly toxic microcystins from lichen-associated cyanobacterium Nostoc sp. strain IO-102-I

The production of hepatotoxic cyclic heptapeptides, microcystins, is almost exclusively reported from planktonic cyanobacteria. Here we show that a terrestrial cyanobacterium Nostoc sp. strain IO-102-I isolated from a lichen association produces six different microcystins. Microcystins were identified with liquid chromatography-UV mass spectrometry by their retention times, UV spectra, mass fragmentation, and comparison to microcystins from the aquatic Nostoc sp. strain 152. The dominant microcystin produced by Nostoc sp. strain IO-102-I was the highly toxic [ADMAdda(5)]microcystin-LR, which accounted for ca. 80% of the total microcystins. We assigned a structure of [DMAdda(5)]microcystin-LR and [d-Asp(3),ADMAdda(5)]microcystin-LR and a partial structure of three new [ADMAdda(5)]-XR type of microcystin variants. Interestingly, Nostoc spp. strains IO-102-I and 152 synthesized only the rare ADMAdda and DMAdda subfamilies of microcystin variants. Phylogenetic analyses demonstrated congruence between genes involved directly in microcystin biosynthesis and the 16S rRNA and rpoC1 genes of Nostoc sp. strain IO-102-I. Nostoc sp. strain 152 and the Nostoc sp. strain IO-102-I are distantly related, revealing a sporadic distribution of toxin production in the genus Nostoc. Nostoc sp. strain IO-102-I is closely related to Nostoc punctiforme PCC 73102 and other symbiotic Nostoc strains and most likely belongs to this species. Together, this suggests that other terrestrial and aquatic strains of the genus Nostoc may have retained the genes necessary for microcystin biosynthesis.     

Identification of a nuclease and host restriction-modification in the unicellular, aerobic nitrogen-fixing cyanobacterium Cyanothece sp.

In the process of developing a gene transfer system for the marine, unicellular, nitrogen-fixing cyanobacterium Cyanothece sp. strain BH68K, two major restriction barriers have been identified. A cell wall-associated nuclease exhibited non-site-specific degradation of covalently closed circular and linear double-stranded DNA molecules, including Cyanothece sp. strain BH68K chromosomal DNA. The nuclease is easily released from intact cells by using water or buffer containing Triton X-100. Nuclease activity was undetectable in cell extracts prepared from water-washed cells. Comparison of the restriction endonuclease susceptibility of Cyanothece sp. strain BH68K DNA to that of Anabaena sp. strain PCC 7120 revealed that these organisms have a nearly identical pattern of restriction and therefore may contain similar systems for DNA methylation. Restriction by DpnI, MboI, and Sau3AI indicated the presence of adenine methylation. Cyanothece sp. strain BH68K cell extracts contain a type II restriction endonuclease, Csp68KI. The activity of Csp68KI was easily detected in cell extracts without extensive purification. Csp68KI is an isoschizomer of AvaII and recognizes the nucleotide sequence 5'-GG(A/T)CC-3'. Cleavage occurs between the guanosine nucleotides producing 3-bp 5' overhang ends.     

Isolation and identification of Streptomyces sp. Act4Zk,a good producer of Staurosporine and some derivatives

In this study, strain Streptomyces sp. Act4Zk was isolated based on a method developed for the isolation of myxobacteria. Due to the low efficiency of the majority of conventional DNA extraction techniques, for molecular identification of the strain Streptomyces sp. Act4Zk, a new technique for DNA extraction of Actinobacteria was developed. In order to explore potential bioactivities of the strain, extracts of the fermented broth culture were prepared by an organic solvent (i.e. ethyl acetate) extraction method using. These ethyl acetate extracts were subjected to HPLC fractionation against standard micro-organisms, followed by LC/MS analysis. Based on morphological, physiological, biochemical and 16S rRNA gene sequence data, strain Streptomyces sp. Act4Zk is likely to be a new species of Streptomyces, close to Streptomyces genecies and Streptomyces roseolilacinus. Antimicrobial assay indicated high antifungal activity as well as antibacterial activity against Mycobacterium smegmatis and Gram-positive bacteria for the new strain. HPLC and LC/MS analyses of the extracts led to the identification of three different compounds and confirmed our hypothesis that the interesting species of the genus Streptomyces being a good producer of staurosporine and some derivatives.