Low-molecular weight natural products display vast structural diversity and have played a key role in the development of novel therapeutics. Here we report the discovery of novel members of the ...aeruginosin family of natural products, which we named varlaxins. The chemical structures of varlaxins 1046A and 1022A were determined using a combination of mass spectrometry, analysis of one- and two-dimensional NMR spectra, and HPLC analysis of Marfey's derivatives. These analyses revealed that varlaxins 1046A and 1022A are composed of the following moieties: 2-
-methylglyceric acid 3-
-sulfate, isoleucine, 2-carboxy-6-hydroxyoctahydroindole (Choi), and a terminal arginine derivative. Varlaxins 1046A and 1022A differ in the cyclization of this arginine moiety. Interestingly, an unusual α-D-glucopyranose moiety derivatized with two 4-hydroxyphenylacetic acid residues was bound to Choi, a structure not previously reported for other members of the aeruginosin family. We sequenced the complete genome of
sp. UHCC 0870 and identified the putative 36 kb varlaxin biosynthetic gene cluster. Bioinformatics analysis confirmed that varlaxins belong to the aeruginosin family of natural products. Varlaxins 1046A and 1022A strongly inhibited the three human trypsin isoenzymes with IC
of 0.62-3.6 nM and 97-230 nM, respectively, including a prometastatic trypsin-3, which is a therapeutically relevant target in several types of cancer. These results substantially broaden the genetic and chemical diversity of the aeruginosin family and provide evidence that the aeruginosin family is a source of strong inhibitors of human serine proteases.
The effect of temperature, light and nutrient composition on morphological traits was determined in seven nostocacean cyanobacteria
(Anabaena planctonica, A. sphaerica
var.
conoidea, A. spiroides, ...Aphanizomenon gracile, Nostoc
sp.,
Scytonema
sp., and
Tolypothrix
sp.). Their morphological variability was high but only some of the features showed changes reflecting varied growth conditions. The frequency of heterocyst occurrence decreased with increasing nitrogen concentration. Within the range studied, the effect of temperature on heterocyst frequency of
Tolypothrix
sp. and planktonic
Anabaena
strains could be fitted by a normal curve with a clear optimum while linear correlation was found in
Aphanizomenon gracile
. T-and S-type branching was observed in both
Scytonema
sp. and
Tolypothrix
sp. strains. T-type branching was found to be markedly dependent on nitrogen concentration. The abundance of necridic cells of
Tolypothrix
sp. increased linearly with temperature and light intensity. Regularity of trichome coiling of
A. spiroides
depended on culture medium, suggesting that nutrient composition may be the main controlling factor. In contrast, the effect of the experimental conditions on the dimensions of vegetative cells and heterocysts was weak. Their variability was markedly higher within each experimental treatment than between treatments.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
1 Department of Applied Chemistry and Microbiology, Viikki Biocenter, PO Box 56, FI-00014 University of Helsinki, Finland
2 University of South Bohemia, Faculty of Biological Sciences, Brani ovská ...31, Cz-370 05 eské Bud jovice, Czech Republic
3 Institute of Hydrobiology, Academy of Sciences of the Czech Republic, Na sádkách 7, Cz-370 05 eské Bud jovice, Czech Republic
4 Public Research Center Gabriel Lippmann, Environment and Biotechnologies Research Unit, 162a avenue de la Faïencerie, L-1511 Luxembourg, Grand-duchy of Luxembourg
5 Institute of Botany, Academy of Sciences of the Czech Republic, Dukelská 145, Cz-379 82 T ebo , Czech Republic
Correspondence Kaarina Sivonen kaarina.sivonen{at}helsinki.fi
The heterocytous cyanobacteria form a monophyletic group according to 16S rRNA gene sequence data. Within this group, phylogenetic and morphological studies have shown that genera such as Anabaena and Aphanizomenon are intermixed. Moreover, the phylogeny of the genus Trichormus , which was recently separated from Anabaena , has not been investigated. The aim was to study the taxonomy of the genera Anabaena , Aphanizomenon , Nostoc and Trichormus belonging to the family Nostocaceae (subsection IV.I) by morphological and phylogenetic analyses of 16S rRNA gene, rpoB and rbcLX sequences. New strains were isolated to avoid identification problems caused by morphological changes of strains during cultivation. Morphological and phylogenetic data showed that benthic and planktic Anabaena strains were intermixed. In addition, the present study confirmed that Anabaena and Aphanizomenon strains were not monophyletic, as previously demonstrated. The evolutionary distances between the strains indicated that the planktic Anabaena and Aphanizomenon strains as well as five benthic Anabaena strains in cluster 1 could be assigned to a single genus. On the basis of the 16S rRNA, rpoB and rbcLX gene sequences, the Anabaena / Aphanizomenon strains (cluster 1) were divided into nine supported subclusters which could also be separated morphologically, and which therefore might represent different species. Trichormus strains were morphologically and phylogenetically heterogeneous and did not form a monophyletic cluster. These Trichormus strains, which were representatives of three distinct species, might actually belong to three genera according to the evolutionary distances. Nostoc strains were also heterogeneous and seemed to form a monophyletic cluster, which may contain more than one genus. It was found that certain morphological features were stable and could be used to separate different phylogenetic clusters. For example, the width and the length of akinetes were useful features for classification of the Anabaena / Aphanizomenon strains in cluster 1. This morphological and phylogenetic study with fresh isolates showed that the current classification of these anabaenoid genera needs to be revised.
Abbreviations: ML, maximum likelihood; MP, maximum parsimony; NJ, neighbour joining; PCA, principal-component analysis
Published online ahead of print on 6 August 2004 as DOI 10.1099/ijs.0.63276-0.
The GenBank/EMBL/DDBJ accession numbers are AJ630408 AJ630458 for the 16S rRNA gene sequences, AJ632022 AJ632070 for the rbcLX gene sequences and AJ628068 AJ628134 for rpoB gene sequences determined in this study.
A table of complete morphological characters is available as supplementary material in IJSEM Online.
The potential for N(2) fixation by heterocystous cyanobacteria isolated from soils of different geographical areas was determined as nitrogenase activity (NA) using the acetylene reduction assay. ...Morphology of cyanobacteria had the largest influence on NA determined under light conditions. NA was generally higher in species lacking thick slime sheaths. The highest value (1446 nmol/h C(2)H(4) per g fresh biomass) was found in the strain of branched cyanobacterium Hassalia (A Has1) from the polar region. A quadratic relationship between NA and biomass was detected in the Tolypothrix group under light conditions. The decline of NA in dark relative to light conditions ranged from 37 to 100 % and differed among strains from distinct geographical areas. Unlike the NA of temperate and tropical strains, whose decline in dark relative to light was 24 and 17 %, respectively, the NA of polar strains declined to 1 % in the dark. This difference was explained by adaptation to different light conditions in temperate, tropical, and polar habitats. NA was not related to the frequency of heterocysts in strains of the colony-forming cyanobacterium Nostoc. Colony morphology and life cycle are therefore more important for NA then heterocyst frequency. NA values probably reflect the environmental conditions where the cyanobacterium was isolated and the physiological and morphological state of the strain.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
1 Department of Botany, Stockholm University, S-10691 Stockholm, Sweden
2 Department of Botany, Faculty of Biological Sciences, University of South Bohemia, eské Bud jovice, Czech Republic
3 ...Institute of Physical Biology, University of South Bohemia, Nové Hrady, Czech Republic
4 Institute of Microbiology, Department of Autotrophic Microorganisms, Academy of Sciences of the Czech Rrepublic, Opatovick Ml n, T ebo , Czech Republic
5 Institute of Ecosystem Study, National Research Council of Italy, via Madonna del Piano 10, 50019 Sesto Fiorentino, Italy
6 PhD School in Science for Conservation of the Cultural Heritage, University of Florence, Italy
7 Institute of Soil Biology, Biology Centre AS CR, v. v. i., Na Sádkách 7, 37005 eské Bud jovice, Czech Republic
8 PhD School in Polar Science, University of Siena, Italy
Correspondence Stefano Ventura stefano.ventura{at}ise.cnr.it
Many cyanobacteria commonly identified as belonging to the genus Nostoc are well-known cyanobionts (symbionts) of a wide variety of plants and fungi. They form symbioses with bryophytes, pteridophytes, gymnosperms and angiosperms that are considerably different in the type of reciprocal interaction between the host and the cyanobiont. The phylogenetic and taxonomic relationships among cyanobionts isolated from different hosts and Nostoc strains isolated from free-living conditions are still not well understood. We compared phylogeny and morphology of symbiotic cyanobacteria originating from different host plants (genera Gunnera , Azolla , Cycas , Dioon , Encephalartos , Macrozamia and Anthoceros ) with free-living Nostoc isolates originating from different habitats. After preliminary clustering with ARDRA (amplified rDNA restriction analysis), phylogeny was reconstructed on the basis of 16S rRNA gene sequences and compared with morphological characterization, obtaining several supported clusters. Two main Nostoc clusters harboured almost all cyanobionts of Gunnera , Anthoceros and of several cycads, together with free-living strains of the species Nostoc muscorum , Nostoc calcicola , Nostoc edaphicum , Nostoc ellipsosporum and strains related to Nostoc commune . We suggest that the frequent occurrence of symbiotic strains within these clusters is explained by the intensive hormogonia production that was observed in many of the strains studied. However, no evidence for discrimination between symbiotic and free-living strains, either by molecular or morphological approaches, could be found. Sequences of Azolla cyanobiont filaments, taken directly from leaf cavities, clustered tightly with sequences from the planktic cyanobacterium Cylindrospermopsis raciborskii , from the benthic Anabaena cylindrica 133 and from Anabaena oscillarioides HINDAK 1984/43, with high bootstrap values. The phylogenetic analysis showed that two distinct patterns of evolution of symbiotic behaviour might exist for the nostocacean cyanobacteria, one leading to symbioses of Nostoc species with a wide variety of plants, the other leading to the association of a unique cyanobacterial type with the water fern Azolla .
Abbreviations: AKLC, akinete-like cells; ARDRA, amplified rDNA restriction analysis; ITS, internal transcribed spacer; ML, maximum-likelihood; MP, maximum-parsimony; NJ, neighbour-joining
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences determined in this study are AM711522 –AM711554.
A supplementary figure showing the ARDRA of symbiotic and free-living cyanobacteria performed on the 16S rRNA gene+ITS region is available with the online version of this paper.
Frequency of heterocytes and nitrogenase activity (NA) under light and dark cultivation conditions was determined in 12 cyanobacterial strains isolated from various soil habitats. In spite of a high ...variability, significant differences in NA among the strains were found in response of light and dark cultivation. Relatively high NA (9.9-15.3 micromol/h C2H4 per g fresh mass) under light conditions and basal NA after 12 h of dark cultivation were detected in Anabaena, Nodularia, Tolypothrix, and 1 of Cylindrospermum strains. On the other hand, significantly lower NA (0.76-5.4 micromol/h C2H4 per g fresh mass) was found under light conditions in Trichormus, Nostoc and another Cylindrospermum strain; the activity completely disappeared after 12 h of dark cultivation. NA values were not directly related to the frequency of the heterocytes. The total NA of cyanobacterial colony was found to be probably independent of the number and/or position of heterocytes. Remarkable differences in NA between strains isolated from cultivated fields and strains originating from natural or non-cultivated soils were found.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Natural substances offer interesting bioactivity patterns including antiproliferative, antioxidant or cytotoxic effects. However, the safety profile of many of them has not been extensively ...determined. In this study, the cytotoxic effect of Aeruginosin-865, resveratrol and capsaicin at different concentrations was tested on normal mouse cells (NIH/3T3) and tumour fibroblasts (WEHI-13VAR) as well as on liver- and kidney-derived cells from fallow deer. A lactate dehydrogenase cytotoxicity assay kit was used to measure cell death in response to treatment with the test substances. It was found that NIH/3T3 cells tolerated Aeruginosin-865 (10-200 μM) and resveratrol (5-100 μM) treatment without any cytotoxic effect, while capsaicin exerted a cytotoxic effect only at the highest tested concentration (200 μM). Mouse fibrosarcoma cells were more sensitive to the cytotoxic effect of all three compounds where Aeruginosin-865 (100-200 μM) and resveratrol (50–100 μM) showed high-dose cytotoxicity and capsaicin showed low- and high-dose cytotoxicity (25 μM and 200 μM). The three tested compounds at the highest concentrations were found to be cytotoxic to both liver- and kidney-derived cells from fallow deer. Overall, the results indicate that the cytotoxic effects of the three tested natural substances on cells derived from fallow deer and mouse tumour fibroblasts differ significantly from those exerted on normal fibroblasts. The results demonstrate the potential of these natural compounds as therapeutic agents and pave the way for future in vivo toxicological investigations.
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