Filamentous cyanobacteria belong to the most prolific producers of structurally unique and biologically active natural products, yet the majority of biosynthetic gene clusters predicted for these multicellular collectives are currently orphan. Here, we present a systems analysis of secondary metabolite gene expression in the model strain Nostoc punctiforme PCC73102 using RNA-seq and fluorescence reporter analysis. Our data demonstrate that the majority of the cryptic gene clusters are not silent but are expressed with regular or sporadic pattern. Cultivation of N. punctiforme using high-density fermentation overrules the spatial control and leads to a pronounced upregulation of more than 50% of biosynthetic gene clusters. Our data suggest that a combination of autocrine factors, a high CO2 level, and high light account for the upregulation of individual pathways. Our overarching study not only sheds light on the strategies of filamentous cyanobacteria to share the enormous metabolic burden connected with the production of specialized molecules but provides an avenue for the genome-based discovery of natural products in multicellular cyanobacteria as exemplified by the discovery of highly unusual variants of the tricyclic peptide microviridin.

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.

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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.

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Summary The object of this study was to compare 4 different methods of oocyte recovery from mares; 1) transvaginal follicle aspiration in vivo; 2) follicle aspiration in vitro; 3) oocyte recovery by isolation of follicles in vitro and 4) follicle scraping in vitro. Oocyte recovery was highest after follicle scraping (71.1%) and follicle isolation and rupture (61.3%). Follicle aspiration in vitro and in vivo yielded oocytes on 31.2% and 19.3% of occasions, respectively. The output of different types of cumulus‐oocyte‐complexes was different among the methods; the portion of compact cumulus‐oocyte‐complexes was significantly higher with follicle scraping (50.7%) and follicle isolation (44.5%) than with aspiration in vivo (31.9%) and in vitro (23.7%). The recovery rate of oocytes from small follicles (<15 mm) was significantly higher than from larger follicles (P < 0.05) using transvaginal follicle aspiration. The proportion of oocytes that were degenerate (exhibited shrunken, dense or visibly damaged ooplasm) ranged from 1.2% after follicle scraping, to 17.2% after aspiration in vivo. These results indicate that, for the recovery of horse oocytes in vitro, follicle scraping and follicle isolation give the highest recoveries of cumulus‐intact oocytes.

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