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Nostoc sp. strain KVJ20 was isolated from the symbiotic organs of the liverwort Blasia pusilla. This cyanobacterium has been shown to have broad symbiotic competence, and bacterial extracts have inhibitory effects on cancer cell lines and microbes. An array of genes for the production of secondary metabolites is present.

Taxonomic compositions of epiphytic bacterial communities in water areas differing in levels of oil pollution were revealed. In total, 82 bacterial genera belonging to 16 classes and 11 phyla were detected. All detected representatives of epiphytic bacterial communities belonged to the phyla Actinobacteria, Bacteroidetes, Planctomycetes, Proteobacteria, Verrucomicrobia, Acidobacteria, Cyanobacteria, Firmicutes, and Fusobacteria and candidate division TM7. The ratio of the phyla in the communities varied depending on the levels of oil pollution. New data on taxonomic composition of uncultivated epiphytic bacterial communities of Fucus vesiculosus were obtained.

Published version. Source at https://doi.org/10.3389/fmicb.2016.01693 Liverwort Blasia pusilla L. recruits soil nitrogen-fixing cyanobacteria of genus Nostoc as symbiotic partners. In this work we compared Nostoc community composition inside the plants and in the soil around them from two distant locations in Northern Norway. STRR fingerprinting and 16S rDNA phylogeny reconstruction showed a remarkable local diversity among isolates assigned to several Nostoc clades. An extensive web of negative allelopathic interactions was recorded at an agricultural site, but not at the undisturbed natural site. The cell extracts of the cyanobacteria did not show antimicrobial activities, but four isolates were shown to be cytotoxic to human cells. The secondary metabolite profiles of the isolates were mapped by MALDI-TOF MS, and the most prominent ions were further analyzed by Q-TOF for MS/MS aided identification. Symbiotic isolates produced a great variety of small peptide-like substances, most of which lack any record in the databases. Among identified compounds we found microcystin and nodularin variants toxic to eukaryotic cells. Microcystin producing chemotypes were dominating as symbiotic recruits but not in the free-living community. In addition, we were able to identify several novel aeruginosins and banyaside-like compounds, as well as nostocyclopeptides and nosperin.

Nostoc punctiforme is a filamentous cyanobacterium capable of forming symbiotic associations with a wide range of plants. The strain exhibits extensive phenotypic characteristics and can differentiate three mutually exclusive cell types: nitrogen-fixing heterocysts, motile hormogonia and spore-like akinetes. Here, we provide evidence for a crucial role of an extracellular metabolite in balancing cellular differentiation. Insertional mutagenesis of a gene of the polyketide synthase gene cluster pks2 led to the accumulation of short filaments carrying mostly terminal heterocysts under diazotrophic conditions. The mutant has a strong tendency to form biofilms on solid surfaces as well as in liquid culture. The pks2(-) strain keeps forming hormogonia over the entire growth curve and shows an early onset of akinete formation. We could isolate two fractions of the wild-type supernatant that could restore the capability to form long filaments with intercalary heterocysts. Growth of the mutant cells in the neighbourhood of wild-type cells on plates led to a reciprocal influence and a partial reconstruction of wild-type and mutant phenotype respectively. We postulate that extracellular metabolites of Nostoc punctiforme act as life cycle governing factors (LCGFs) and that the ratio between distinct factors may guide the differentiation into different life stages.

Phylogenetic relationships between Nostoc cyanobionts in the lichen genus Pannaria were studied to evaluate their correlation to geography, habitat ecology, and other patterns previously reported. The 16S rRNA gene sequences of a total of 37 samples of 21 Pannaria species from seven countries from the Northern and Southern hemispheres were analyzed and compared with 69 free-living and symbiotic cyanobacterial strains. The sequences from Pannaria were distributed throughout a branch of Nostoc sequences previously called ""the Nephroma guild,"" and within two subgroups from another branch, referred to as the ""Peltigera guild,"" although there was a gradual transition between the two major groups. There is a more diverse pattern of relationships between Nostoc sequences from bipartite versus tripartite lichen species in Pannaria, compared with other well-studied genera, such as Nephroma and Peltigera. Cyanobionts from several tripartite Pannaria species from the Southern Hemisphere and corticolous bipartite species from both hemispheres were grouped together. Four sequences of Pannaria and Pseudocyphellaria cyanobionts from rocks in the Chilean Juan Fernandez Islands were nested within corticolous cyanobionts, whereas the terricolous ""Pannaria sphinctrina clade"" was placed with other terricolous strains. The cluster patterns derived from phylogenetic analysis were partly reflecting lichen taxonomy, in two groups of lichen species, possibly indicating coevolution. The phylogram partly also reflected lichen ecology. Three Pannaria species have very different cyanobiont strains when they grow in different habitats.

Nostoc punctiforme is a versatile cyanobacterium that can live either independently or in symbiosis with plants from distinct taxa. Chemical cues from plants and N. punctiforme were shown to stimulate or repress, respectively, the differentiation of infectious motile filaments known as hormogonia. We have used a polyketide synthase mutant that accumulates an elevated amount of hormogonia as a tool to understand the effect of secondary metabolites on cellular differentiation of N. punctiforme. Applying MALDI imaging to illustrate the reprogramming of the secondary metabolome, nostopeptolides were identified as the predominant difference in the pks2(-) mutant secretome. Subsequent differentiation assays and visualization of cell-type-specific expression of nostopeptolides via a transcriptional reporter strain provided evidence for a multifaceted role of nostopeptolides, either as an autogenic hormogonium-repressing factor or as a chemoattractant, depending on its extracellular concentration. Although nostopeptolide is constitutively expressed in the free-living state, secreted levels dynamically change before, during, and after the hormogonium differentiation phase. The metabolite was found to be strictly down-regulated in symbiosis with Gunnera manicata and Blasia pusilla, whereas other metabolites are up-regulated, as demonstrated via MALDI imaging, suggesting plants modulate the fine-balanced cross-talk network of secondary metabolites within N. punctiforme.

Nostoc punctiforme is a filamentous cyanobacterium capable of forming symbiotic associations with a wide range of plants. The strain exhibits extensive phenotypic characteristics and can differentiate three mutually exclusive cell types: nitrogen-fixing heterocysts, motile hormogonia and spore-like akinetes. Here, we provide evidence for a crucial role of an extracellular metabolite in balancing cellular differentiation. Insertional mutagenesis of a gene of the polyketide synthase gene cluster pks2 led to the accumulation of short filaments carrying mostly terminal heterocysts under diazotrophic conditions. The mutant has a strong tendency to form biofilms on solid surfaces as well as in liquid culture. The pks2(-) strain keeps forming hormogonia over the entire growth curve and shows an early onset of akinete formation. We could isolate two fractions of the wild-type supernatant that could restore the capability to form long filaments with intercalary heterocysts. Growth of the mutant cells in the neighbourhood of wild-type cells on plates led to a reciprocal influence and a partial reconstruction of wild-type and mutant phenotype respectively. We postulate that extracellular metabolites of Nostoc punctiforme act as life cycle governing factors (LCGFs) and that the ratio between distinct factors may guide the differentiation into different life stages.

The ability of cyanobacteria to produce the phytohormone indole-3-acetic acid (IAA) was demonstrated. A colorimetric (Salkowski) screening of 34 free-living and symbiotically competent cyanobacteria, that represent all morphotypes from the unicellular to the highly differentiated, showed that auxin-like compounds were released by about 38% of the free-living as compared to 83% of the symbiotic isolates. The endogenous accumulation and release of IAA were confirmed immunologically (ELISA) using an anti-IAA antibody on 10 of the Salkowski-positive strains, and the chemical authenticity of IAA was further verified by chemical characterization using gas chromatography-mass spectrometry in Nostoc PCC 9229 (isolated from the angiosperm Gunnera) and in Nostoc 268 (free-living). Addition of the putative IAA precursor tryptophan enhanced IAA accumulation in cell extracts and supernatants. As the genome of the symbiotically competent Nostoc PCC 73102 contains homologues of key enzymes of the indole-3-pyruvic acid pathway, a transaminase and indolepyruvate decarboxylase (IpdC), the putative ipdC gene from this cyanobacterium was cloned and used in Southern blot analysis. Out of 11 cyanobacterial strains responding positively in the Salkowski/ELISA test, ipdC homologues were found in 4. A constitutive and possibly tryptophan-dependent production of IAA via the indole-3-pyruvic acid pathway is therefore suggested. The possible role of IAA in cyanobacteria in general and in their interactions with plants is discussed.

Cyanobacteria are an excellent source of bioactive natural products that can be used in the development of new medicinal drugs. The cyanobacterial genus Nostoc have proven to be prolific producers of molecules with exciting bioactivities, including anti-bacterial, anti-fungal, and anti-cancerous. This feature combined with the Nostoc’s complex life cycle and sizeable genomes make them interesting targets for bioprospecting. During this thesis, a typical natural product discovery pipeline was carried out to investigate some of the genomic and metabolomic characteristics of two Nostoc sp. strains. The pipeline included prediction of the biosynthetic potential, liquid extraction of metabolites using a variation of solvents, reverse-phase fractionation, bioassay to test for bioactivity, and the use of reverse-phase UHPLC-MS² and an informatic tool for dereplication and molecular networking. Some clear differences between the strains were observed in the prediction of metabolite production and in the extracted metabolites. Anti-bacterial activity was observed in extract fractions of both strains, and the dereplication process resulted in the discovery of a congener of the earlier described compound hapalosin which has been shown to reverse multi-drug resistance in cancer cells.

Hydrocarbon pollution in marine environments present an acute problem which is aggravated by cold temperatures. This is especially relevant in important environmental and economic northern regions such as the Barents Sea. The Barents Sea region has become the focus of oil industries from Russia and Norway, incrementing the risk of hydrocarbon pollution. Bioremediation is a cost-effective and environmentally sound method to remove hydrocarbon pollution. To study the bioremediation potential of native bacteria from a chronically oil polluted region of the Barents Sea a study was done in the Murmansk seaport (Kola Bay, 68°58′00′′ N, 33°05′00′′ E) analyzing the composition of the bacterial community. This present work aims to do a deeper study of that bacterial community through a metagenomic fosmid library, a selective hydrocarbon-rich medium and bioinformatic analysis. A metagenomic fosmid library was constructed with the environmental DNA, the fosmid clones were transformed in Escherichia coli cells and cultivated in minimal media with concentrations 0.05%, 0.5% and 1% of diesel or crude oil. Ten colonies were selected, sequenced, and subsequently analyzed with the software Geneious 2020.2.2. BLAST searches and other bioinformatic tools were conducted in every Open Reading Frame of the 10 colonies, revealing new links between native bacteria and hydrocarbon degradation as well as promising enzymes for hydrocarbon bioremediation. This work sets the ground for further studies on functional metagenomic analyses with fosmid libraries and further studies with the novel bacterial species and enzymes linked to hydrocarbon biodegradation.

Widespread mortality and disease are recognized as growing problems in benthic marine coral and sponge communities across the globe and despite being extensively studied, in most cases the etiology of disease remains unknown. Since 2011, sponge communities in freshwater Lake Baikal, Siberia, Russia have been affected by an unknown agent of disease, which has been causing an array of physical disease manifestations. Unusual microbial patterns have also been noted in the lake, including cyanobacterial bloom development and cyanobacterial biofilm growth on sponges, which have concurrently occurred with the overall decline of sponge communities. We used UPLC-HR-MS/MS and 16S rRNA sequencing to profile healthy and diseased sponges in order to elucidate the role of cyanobacteria in the development of sponge disease. Diseased specimens from the four sponge genera found in the littoral zone, encompassing a broad range of disease manifestations from violet-brown spots and bacterial biofilms to bleaching, were investigated. Within the four sponge genera, we discovered slight intergenera differences between microbial community composition (at the phylum and cyanobacterial genus levels) and metabolic output in the healthy representatives. As expected, most diseased sponges showed differences in their microbial community composition (at the phylum and cyanobacterial genus levels) and metabolic profiles in comparison to their healthy counterparts, and some connections could be drawn within sponges exhibiting similar disease symptoms. Though the association between cyanobacteria and disease etiology was not supported by the results of the metabolomic and metagenomic profiling and cyanotoxins were not discovered, this thesis provides a solid foundation for the ongoing, more in-depth study of the changes occurring in the Lake Baikal littoral sponge community.

Cyanobacteria are a unique source of natural products, where most of them are synthesized by non-ribosomal peptide synthase, polyketide synthase, or a hybrid of these pathways. The identification of such biosynthetic genes responsible for the production of secondary metabolites is still a relatively unexplored area, and it remains many natural products for which a biosynthetic origin is unknown. Secondary metabolites from marine cyanobacteria have gained much attention the last decades, however few comprehensive studies on secondary metabolites and their biosynthetic gene clusters from terrestrial cyanobacteria have been conducted. Three terrestrial Nostoc spp. KVJ20, KVJ2, and KVJ10 were recently sequenced which allowed us to conduct genome-wide predictions by AntiSMASH of their potential to produce secondary metabolites. These strains were subjected to various cultivation conditions including nutrient limitations and competition. Gene expression analysis by RT-qPCR of predicted gene clusters and the production of secondary metabolites by UPLC-HR-MS were conducted. Analysis of gene expression patterns revealed higher expression of several NRPS, PKS and RiPP genes in nutrient-deprived media, as well as confirming the present known function of the housekeeping genes; NifH, GvpC, and PilT. Most of the secondary metabolites found by UPLC-HR-MS were not identified, however a variety of Nostocyclopeptides, Suomilide/Banyaside-like peptides, Anabaenopeptins, as well as Aeruginosin, Hapalosin, and Nosperin were recorded from extracts of the respective strains. The results from this thesis give valuable knowledge for further cultivation of terrestrial cyanobacteria, with the purpose of awaking cryptic gene clusters and identifying novel secondary metabolites. We have also suggested conditions most suitable for enrichment for identified compounds.