Browsing by Subject "cyanobacteria"

Aims of this study. Previous studies have shown cyanobacterial dominance and harmful cyanobacterial blooms to increase due to recent climate warming. The increase of aggressively blooming species and toxin-producing strains of cyanobacteria has been predicted to further increase in the future. However, information on the response of cyanobacteria communities to environmental forcing in the Arctic region – which is experiencing warming at over twice the rate compared to the global average – has been insufficient. Thus, it is crucial to study how algal and cyanobacterial communities have developed after industrialization to better understand and predict future trends of subarctic algal communities as well as changes within cyanobacteria communities experiencing environmental forcing. This study aims to provide information on the effect of recent climate warming and lake browning on algal communities in subarctic lakes, with a special focus on cyanobacteria and cyanotoxins. Materials and methods. Modern and historical primary producer group abundances of 23 subarctic lakes located on an ideal temperature and vegetation gradient were studied using sedimentary algal pigments as a proxy. The top-bottom method was used to study both changes within algal communities during the last ca. 150 years and the broader trends in algal communities of subarctic lakes. Pigment data was analyzed together with environmental data using ordination analyses (principal component analysis (PCA) and redundancy analysis (RDA)) as well as other statistical analyses in order to determine possible trends of change and to reveal the environmental variables that have the strongest impact on cyanobacterial abundance. Results and conclusions. Algal communities have changed during the last ca. 150 years and show a general trend of increased primary production as well as lake browning in the spruce, pine and birch (SPB) vegetation zone. Siliceous algae generally dominate modern algal communities, and relative abundances of cyanobacteria have declined throughout the vegetation gradient. Within the Barren (Ba)- and mountain birch woodland (MBW) vegetation zones, cyanobacteria communities show a marked decline in the abundance of assumed benthic species based on pigment data, and low abundances of planktic picocyanobacteria. However, due to climate warming and lake browning, abundances of cyanobacteria have increased in several sites within the SPB vegetation zone and are suspected to indicate an increase of harmful planktic species. The most significant environmental variables controlling the abundance of cyanobacteria were total phosphorus, temperature and the amount of organic matter. The results highlight the urgent need to mitigate climate warming in order to preserve the unique biota and characteristics of Arctic and subarctic lake ecosystems, and to prevent the possible harmful increase of cyanotoxins in these sensitive ecosystems.

Cyanobacteria are phototrophic organisms. They usually occur in water but many species also live in terrestrial habitats, e.g. in symbiotic relationships with fungus. Inorganic phosphorus (Pi) is usually considered to be a limiting factor for the growth of cyanobacteria living in water, since cyanobacteria can use only Pi as a direct source of phosphorus. It has been shown that cyanobacteria have pho-regulon similar to that of Escherichia coli. Pho-regulon can transport and assimilate inorganic phosphate. Cyanobacteria usually produce a wide range of bioactive compounds, which can e.g. be toxic or prevent growth of other bacteria, fungi or yeast. Many of these compounds are produced by non-ribosomal peptide synthetases (NRPS). The aim of this study was to investigate changes in Anabaena sp. 90 proteome, and differences in amounts of bioactive compounds produced by the strain, while growing it in media with high (5,5 mg/l) or low (0,05 mg/ml) Pi concentration. Before moving the culture into two different Pi media, phosphorus storages of the culture were emptied by growing the strain in the media without Pi. In this study, 2D differential gel electrophoresis (2D-DIGE) coupled with LC/MS was used to study proteomes of the organism. Bioactive compounds were also analyzed by LC/MS. Anabaena sp. 90 was chosen because of its fully sequenced and annotated genome. The strain has been found to produce microcystins, anabaenopeptins and anabaenopeptilides. Eleven protein spots with significantly increased or decreased protein quantities were identified in the low Pi media. Ten of them were identified as proteins, which participate in bacterial stringent response. Stringent response is activated when culture is achieving the stationary phase. These stringent response proteins participate in the amino-acid metabolism and translation. One of the proteins was shown to be a ribosomal protein. In addition, the identified proteins included ribulose-bisphosphate carboxylase oxygenase (RuBisCO), which had a significantly lower concentration in the cells in low-phosphorous media. There were no significant differences in amounts of bioactive compounds when growing the culture in low and high Pi media. More replicates could be used, when the study of bioactive compounds is repeated.

Cyanobacteria are an important part of the phytoplankton community and aquatic ecosystems. Cyanobacteria can form large mass occurrences, i.e. blooms, which can be toxic or cause other harm. Research and monitoring of cyanobacteria has been based on microscopy analysis. However, molecular-based methods, such as 16S rRNA sequencing are replacing microscopy analyses in the near future. The Finnish Environment Institute has stated that molecular methods are part of environmental monitoring before 2030. In this Master’s thesis the aim was to determine whether conventional microscopy analyses and 16S rRNA sequencing differ when comparing nano- and micro-sized cyanobacteria. The material was collected from a laboratory experiment of the Finnish Environment Institute’s (SYKE) MiDAS project, which was conducted in the summer of 2020. The results of the microscopy and 16S rRNA analyses differed from each other. The relative abundances of the cyanobacteria genera differed between sample types. Microscopy analyses estimated that the alpha diversity was higher compared to the results of the sequencing analyses. The main reason for the difference between the types of analyses was due to the differences in cyanobacteria belonging to the order of Synechococcales. Some of the Synechococcales species were observed only by the sequencing analyses, e.g. Snowella and some of the Synechococcales species were only observed by the microscopy analyses, e.g. Romeria and Woronichinia. It was observed that both methods are prone to identification errors. The differences between the 16S rRNA sequencing and the microscopy analyses are vastly different. It may affect on the review of long-term data of the phytoplankton community. Therefore, it is important to examine the differences between the types of analyses. Studying the dissimilarities between the types of analyses should be focused on the research of the small cell-sized colonial cyanobacteria, i.e. the species of Chroococcales and Synechococcales.

Biochar is a product from the pyrolysis of plant derived-biomass and it is intended to be applied to soil given its potential of carbon sequestration and soil fertility improvement. Some studies also suggest that increasing application rate of biochar has a positive feedback on biological nitrogen fixation (BNF) and on soil microbial biomass. However, these effects are not well known for boreal forests. The purpose of this study was to evaluate the effects of different biochar application rates: 0 t ha-1, 5 t ha-1 and 10 t ha-1 on BNF, on microbial biomass carbon and nitrogen (MBC and MBN), and on moss biomass. The field experiment was established in Juupajoki, Southern Finland in young Scots pine stands. The stands were amended with biochar one year before the measurements took place. BNF was determined using acetylene reduction assay (ARA), and microbial biomass was estimated using chloroform fumigation-direct extraction (CFDE). The microbial biomass samples were incubated at the temperatures: 10 °C, 15 °C and 20 °C. Biochar amendment raised soil pH, whereas no differences were verified for BNF, MBC, MBN, nor for moss biomass. There was, however, variation in the response of N fixation to incubation temperature, and variation in the response of MBC and MBN to the time of measurement. Observed changes in pH are often likely to justify variations in the rates of BNF and MB, however in this study they were not shown to be of significance. It is possible, however that biochar will have a positive effect on soil vegetation as it is incorporated into the soil in the long-term. Although this study focuses on BNF and MB, the findings may well have a bearing on the use of biochar as a tool for C sequestration, since amendment with biochar was demonstrated as neither beneficial nor harmful to the soil biota.

The Baltic Sea is a unique and delicate brackish water ecosystem with high primary productivity driven by oceanic biogeochemical cycles of oxygen, iron, silicon, nitrogen and phosphorus. Elevated anthropogenic nutrient loading into the Baltic ecosystem has resulted in a large-scale increase in destructive cyanobacterial blooms in the open Baltic Sea over the past century. The toxic cyanobacterium Nodularia spumigena is a major component of surface blooms in the open Baltic Sea and continues to bloom even after the depletion of phosphate from the surrounding waters. This phenomenon has been attributed to an ability to scavenge phosphorus from recalcitrant sources. However, the exact phosphorus species that sustain N. spumigena growth in the Baltic Sea remain largely unknown. Here, I employ a comparative genomics approach to determine the evolutionary dynamics of phosphorus scavenging in eight strains of N. spumigena and predict the range of phosphorus sources that may support their growth. Then, I test these predictions by growing six strains of N. spumigena on a number of potentially bioavailable phosphorus sources. Among the phosphorus scavenging genes identified by the genomic analysis, putative pathways for the enzymatic degradation of phytic acid, phosphite, and phosphonates were present and highly conserved in the species. Subsequent growth experiments demonstrated that the organism may grow using phytic acid and phosphite, as well as the phosphonates methylphosphonic acid, ethylphosphonic acid, and nitrilotris(methylenephosphonic acid), as sole phosphorus sources. These results indicate that N. spumigena blooms may be supported by several phosphorus sources previously not known to contribute to eutrophication in the Baltic Sea. While additional growth experiments and further research on the environmental prevalence of these compounds are necessary, the findings presented in this study expand our knowledge of how N. spumigena dominates phytoplankton blooms in a phosphorus-scarce environment and may help to inform future eutrophication mitigation efforts in the Baltic Sea region.

Mycosporines and mycosporine-like amino acids (MAAs) are small-molecules that provide UV protection in a broad range of organisms. Cyanobacteria produce a diverse set of MAA chemical variants, many of which are glycosylated. Even though the biosynthetic pathway for the production of a common cyanobacterial MAA, shinorine, is known, the biosynthetic origins of the glycosylated variants remains unclear. In this work, bioinformatics analyses were performed to catalogue the genetic diversity encoded in the MAA gene clusters in cyanobacterial genomes and identify a set of enzymes that might be involved in MAA biosynthesis. A total of 211 cyanobacterial genomes were found to contain the MAA gene cluster, with six containing glycosyltransferase genes within the gene cluster. Afterwards, 38 strains from the University of Helsinki Culture Collection were tested for the production of MAAs using QTOF-LC/MS analyses. This resulted in the identification of several novel glycosylated MAA chemical variants from Nostoc sp. UHCC 0302, which contained a 7.4 kb MAA biosynthetic gene cluster consisting of 7 genes, including two for glycosyltransferases and one for dioxygenase. Heterologous expression of this gene cluster in Escherichia coli TOP10 resulted in the production of a glycosylated porphyra-334 variant of 509 m/z by the transformant cells, showing that colanic acid biosynthesis glycosyltransferases can catalyse the addition of hexose to MAAs. These results suggested a biosynthetic route for the production of glycosylated MAAs in cyanobacteria and allowed to propose a putative role for dioxygenases in MAA biosynthesis. Further characterization of additional glycosyltransferases is necessary to improve our understanding of glycosylated MAA biosynthesis and functionality, which could be applied to large scale processes and be used in industrial applications.

Antibiotic resistance is a worldwide problem and it threatens the prevention and treatment of infections caused by different pathogens. All living organisms produce natural products including ribosomal peptides with great variety. They are widely distributed in nature and they are playing more significant role in the search of new antimicrobial compounds used as therapeutical agents. Bacteria are a prolific source of peptides many of which are antimicrobial and microbial genomes are widely believed to encode new antimicrobial peptides. Genome mining has expanded the number of families of ribosomally synthesized natural products in recent years. These In silico approaches together with molecular biology and chemical analysis aim to identify novel compounds. In this study an unknown cyanobactin-like gene cluster was discovered by genome mining from genomes of cyanobacteria and also other bacteria. The aim of this work was to study the occurrence of the gene clusters in various bacterial genomes and the structures of novel peptides. The active biosynthesis of these peptides was tested by LCMS- and Q-TOF -analyses based on bioinformatic predictions. The production of the predicted peptides was also tested with stable sulphur isotope labelling. The aim was also to clone the genes needed for peptide biosynthesis into E. coli and to study antimicrobial activities of these peptides. Bioinformatic analyses suggested that the gene clusters encoded 1–8 precursor peptides together with protease. The precursor peptides had conserved leader sequence (LPxQxxPVxR) and a highly variable core sequences, often encoding an even number of cysteines. The mature peptide is eventually formed from core sequence through post-translational changes in the precursor peptide. The gene cluster was present in 38 bacterial genomes representing a diverse selection of bacterial phyla including cyanobacteria, proteobacteria, actinobacteria, bacteroidetes, firmicutes and planctomycetes. Analyses of the precursor peptide core regions suggested that the products are 8–131 amino acids in length. These peptides could be divided into two groups based on their structures: They form a selection of disulphide-bridge stabilized peptides with 2–5 disulphide-bridges as well as short cationic peptides with an ?-helical structure. Surprisingly, these types of peptides are common in eukaryotes and part of the innate immune system displaying potent antimicrobial properties but very rarely reported for bacteria. The peptides predicted from bioinformatic analysis were detected from Pseudanabaena sp. PCC 6802 using a combination of molecular biology and structural chemistry. Heterologous expression of the gene cluster from Pseudanabaena sp. PCC 6802 in E. coli confirmed that the gene cluster is active. A set of short cationic synthetic peptides with ?-helical structure predicted from Oscillatoria sp. PCC 10802, Dickeya zeae Ech1591, Vibrio nigripulchritudo SOn1, Agarivorans albus MKT 106, Roseibium sp. TrichSKD4 and Yersinia frederiksenii ATCC 33641 were shown to have potent antimicrobial activity between 0.8–100 ?g/ml. These findings prove that predicted cysteine containing peptides are produced by bacteria and some peptides from this novel family have antimicrobial activity, which might pave the way for new possible drugs derived from natural products.

Bacteria are a great source of natural products with complex chemical structures and diverse biological activities. Many have therapeutic properties and half of drugs in clinical use today are derived directly or indirectly from natural products. The pharmaceutical industry stopped investing in drug development from natural resources, due to perceived limitations in chemical space, and difficulties in rediscovery of known compounds and in obtaining sufficient quantities of natural products for clinical trials. There is now renewed interest in natural products as drug leads driven by technological advances in genome sequencing and analytical chemistry. Cyanobacteria produce a variety of natural products with therapeutic potential. Muscoride A is an unusual peptide alkaloid produced by a terrestrial freshwater cyanobacterium with reported antimicrobial activity. The aim of this study was to characterize the biosynthetic origin and biological activity of muscoride A. I identified the 12.7 kb muscoride (mus) biosynthetic gene cluster from a draft genome of Nostoc sp. PCC 7906 using bioinformatics analysis. The mus biosynthetic gene cluster encoded enzymes for the heterocyclization, oxidation and prenylation of a precursor protein. Comparative genomics identified a mus biosynthetic gene cluster in the unpublished draft genome of Nostoc sp. UHCC sp. 0398 encoding a novel muscoride. This novel muscoride, muscoride B, was detected from Nostoc sp. UHCC 0398 based on this analysis. Muscoride B was purified using solid phase extraction and high-performance liquid chromatography and the chemical structure was verified by combining nuclear magnetic resonance and mass spectrometry data. Furthermore, the function and evolutionary history of the muscoride prenyltransferases were studied. A significant finding was that the biosynthetic pathway encodes two regiospecific prenyltransferases, catalyzing the C- and N-terminal prenylation of muscoride. An antimicrobial activity screening showed that muscoride B had antimicrobial activity against Bacillus cereus. Here I report the discovery of the muscoride biosynthetic pathway and the discovery of a novel antimicrobial peptide from cyanobacteria through genome mining. The results show that the variant is a novel muscoride, a linear bis-prenylated polyoxazole pentapeptide with antimicrobial activity.

Lantibiotics are a subgroup of bacteriocins, produced by Gram-positive bacteria to inhibit the growth of closely related strains. They are used as food preservatives e.g. nisin, and some are in clinical trials, e.g. duramycin A and microbisporicin. Cinnamycin is a 19 amino acid lantibiotic that inhibits the growth of Gram-positive rods. Recent work suggests that cyanobacteria might be able to make variants of cinnamycin. Here I determined the product of a cinnamycin biosynthetic pathway present in the genomes of a benthic cyanobacteria. The genome mining analysis demonstrated that three cyanobacterial strains and seven actinobacterial strains contained the genes responsible for the production of cinnamycin. Cinnamycin variants were detected from cyanobacteria Oscillatoria sp. PCC 10802 and actinobacteria Streptomyces roseoverticillatus DSM 40845, respectively. Oscillatoria sp. PCC 10802 produced a cinnamycin variant named oscillamycin, with mass of 1966.86 Da. Stable nitrogen (15N) and sulphur (34S) isotope labeling of the cyanobacterium indicated that the oscillamycin contains 3 sulfur atoms and 23 nitrogen atoms. However, the mass of oscillamycin was 16 units bigger than the bioinformatic predictions. LC-MS analysis suggested that the oscillamycin contains a hydroxyl-proline in addition to hydroxyl aspartic acid. The oscillamycin gene cluster was cloned and successfully expressed in Escherichia coli BL21. Small amounts of oscillamycin (0.25 µg) were purified from Oscillatoria sp. PCC 10802 and showed tentative antimicrobial activity against Bacillus subtilis HAMBI 251. This study demonstrated that cyanobacteria and actinobacteria share a lantibiotic gene cluster and that the lantibiotic produced differed in just four amino acids. The phylogenetic analysis suggested that the cinnamycin gene cluster was transferred from actinobacteria to cyanobacteria by an ancient horizontal gene transfer event. This study expands the chemical diversity of cinnamycin variants. This is the first report of a lantibiotic from cyanobacteria suggesting that cyanobacteria might be a novel source of antibiotics, which could be useful in addressing the antibiotic resistance issue.

Natural products have enormous structural and chemical diversity and are either the source or direct inspiration for many drugs in use today. Cyanobacteria are prolific producers of complex natural products with serine protease inhibiting activity. Many of these natural products are the product of non-ribosomal peptide synthetase (NRPS) modular enzyme complexes. Suomilide is a complex tetrapeptide produced by strains of the benthic cyanobacterium Nodularia sphaerocarpa. It has a highly complicated structure and contains an unusual azabicyclononane moiety, a methylglyceric acid, a xylose unit with hexanoic acids and a terminal 1-amidino-3-(2-aminoethyl)-3-pyrroline moiety. Suomilide inhibits thrombin, plasmin and trypsin in low micro-molar concentrations. The biosynthetic of this unusual glycoside remain unclear. However, suomilide is long predicted to be part to the aeruginosin family of protease inhibitors. A 5.4 Mb draft genome of Nodularia sphaerocarpa HKVV was obtained in order to identify the suomilide biosynthetic. The 43.7 kb suomilide gene cluster was identified on a single contig by performing tBLASTn searches on the draft genome of Nodularia HKVV using aerDEF genes from aeruginosins gene cluster as query. This gene cluster encodes 27 genes including two complex NRPS enzymes and a set of tailoring enzymes for the assembly of suomilide. The suomilide gene cluster shares extensive homology to known aeruginosin gene clusters including two aerB and aerG genes encoding NRPS enzymes, 12 genes (aerC, aerD, aerE, aerF, aerI, aerK, two copies of aerN and four copies of aerH) encoding for the enzymes responsible for synthesis of precursor non-proteinogenic amino acids and 13 other tailoring enzymes. The suomilide gene cluster was much larger and encoded a greater number of biosynthetic enzymes reflecting the structural complexity of suomilide. We identified 10 aeruginosin gene clusters and 2 suomilide gene clusters from 12 strains of cyanobacteria by genome mining. Bioinformatics analyses suggested these gene clusters encoded an unanticipated chemical diversity of aeruginosins and suomilides. LC-MS and Q-TOF analysis detected aeruginosins or suomilide variants from 12 of the 15 strains. Surprisingly, inhibition assays with the crude extracts using all three isoforms of human trypsin suggest that these compounds may have potent and selective inhibition of human trypsin isoforms. Further work is required to prove that suomilide alone can carry out selective inhibition of trypsin isoforms or is it a result of synergism between the compounds produce by cyanobacteria. Phylogenetic analysis demonstrated that the aeruginosin evolved through the acquisition of multiple loading mechanisms and tailoring enzymes through horizontal gene transfers. Our results support the hypothesis that suomilides are a part of aeruginosin family as they are made through the same genetic pathway, however have gained a greater degree of structural diversity due to the acquisition of tailoring enzymes. These results together suggest that cyanobacteria produce an unexpected wealth of complex natural products belonging to the aeruginosin family and that some of these may be potent and selective inhibitors of isoforms of human trypsin.