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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.

Photosynthesis is readily impaired by high light (HL) levels. Photosynthetic organisms have therefore evolved various mechanisms to cope with the problem. Here, we have dramatically enhanced the light tolerance of the cyanobacterium Synechocystis by adaptive laboratory evolution (ALE). By combining repeated mutagenesis and exposure to increasing light intensities, we generated strains that grow under extremely HL intensities. HL tolerance was associated with more than 100 mutations in proteins involved in various cellular functions, including gene expression, photosynthesis and metabolism. Co-evolved mutations were grouped into five haplotypes, and putative epistatic interactions were identified. Two representative mutations, introduced into non-adapted cells, each confer enhanced HL tolerance, but they affect photosynthesis and respiration in different ways. Mutations identified by ALE that allow photosynthetic microorganisms to cope with altered light conditions could be employed in assisted evolution approaches and could strengthen the robustness of photosynthesis in crop plants. Iterating mutagenesis and exposure to increasing light dramatically enhanced the light tolerance of a Synechocystis cyanobacterium strain. This involved over 100 mutations grouped around five haplotypes, as well as putative epistatic interactions.

The frequent production of the hepatotoxin microcystin (MC) and its impact on the lifestyle of bloom-forming cyanobacteria are poorly understood. Here, we report that MC interferes with the assembly and the subcellular localization of RubisCO, in Microcystis aeruginosa PCC7806. Immunofluorescence, electron microscopic and cellular fractionation studies revealed a pronounced heterogeneity in the subcellular localization of RubisCO. At high cell density, RubisCO particles are largely separate from carboxysomes in M. aeruginosa and relocate to the cytoplasmic membrane under high-light conditions. We hypothesize that the binding of MC to RubisCO promotes its membrane association and enables an extreme versatility of the enzyme. Steady-state levels of the RubisCO CO2 fixation product 3-phosphoglycerate are significantly higher in the MC-producing wild type. We also detected noticeable amounts of the RubisCO oxygenase reaction product secreted into the medium that may support the mutual interaction of M. aeruginosa with its heterotrophic microbial community.

The frequent production of the hepatotoxin microcystin and its impact on the life-style of bloom-forming cyanobacteria are poorly understood. Here we report that microcystin interferes with the assembly and the subcellular localization of RubisCO, inMicrocystis aeruginosaPCC7806. Immunofluorescence, electron microscopic and cellular fractionation studies revealed a pronounced heterogeneity in the subcellular localization of RubisCO. At high cell density, RubisCO particles are largely separate from carboxysomes inM. aeruginosaand relocate to the cytoplasmic membrane under high-light conditions. We hypothesize that the binding of microcystin to RubisCO promotes its membrane association and enables an extreme versatility of the enzyme. Steady-state levels of the RubisCO CO2fixation product 3-phosphoglycerate are significantly higher in the microcystin-producing wild type. We also detected noticeable amounts of the RubisCO oxygenase reaction product secreted into the medium that may support the mutual interaction ofM. aeruginosawith its heterotrophic microbial community.

International audience; The cyanobacterial genus Microcystis is known to produce an elaborate array of structurally unique and biologically active natural products, including hazardous cyanotoxins. Cytotoxic aeruginoguanidines represent a yet unexplored family of peptides featuring a trisubstituted benzene unit and farnesylated arginine derivatives. In this study, we aimed at assigning these compounds to a biosynthetic gene cluster by utilizing biosynthetic attributes deduced from public genomes of Microcystis and the sporadic distribution of the metabolite in axenic strains of the Pasteur Culture Collection of Cyanobacteria. By integrating genome mining with untargeted metabolomics using liquid chromatography with mass spectrometry, we linked aeruginoguanidine (AGD) to a nonribosomal peptide synthetase gene cluster and coassigned a significantly smaller product to this pathway, microguanidine (MGD), previously only reported from two Microcystis blooms. Further, a new intermediate class of compounds named microguanidine amides was uncovered, thereby further enlarging this compound family. The comparison of structurally divergent AGDs and MGDs reveals an outstanding versatility of this biosynthetic pathway and provides insights into the assembly of the two compound subfamilies. Strikingly, aeruginoguanidines and microguanidines were found to be as widespread as the hepatotoxic microcystins, but the occurrence of both toxin families appeared to be mutually exclusive. M icrocystis is a dominant bloom-forming cyanobacterium occurring in temperate freshwater ecosystems. 1 The genus is infamous for the production of the well-known hepatotoxin microcystin. 2 Both blooms and toxins cause ecosystem disturbance and public health threats and constitute a growing concern in the frame of freshwater eutrophication and global warming. Microcystis has also been described as a producer of a multitude of bioactive natural products, some of interest for biotechnological and pharmaceutical application. 3−5 Cytotoxic aeruginoguanidines (AGDs) represent one of the most remarkable families of compounds described for Microcystis. 6 The three AGD congeners reported for strain Microcystis aeruginosa NIES-98 feature highly unprecedented characteristics such as a 1-(4-hydroxy-3-hydroxymethyl)-phenyl-1-hydroxy-2-propylamine-4′,3′,1-triO -sulfate (Hphpa trisulfate) moiety, along with geranylation and prenylation of arginines (Figure 1A). While bloom-forming Microcystis belong to the most intensively studied cyanobacteria, AGDs were reported only twice from a bloom in Czech Republic and an isolate in Brazil, 7,8 never from any other cyanobacteria. Their intricate features confine AGDs into a unique compound family. 3 Our recent genomic analysis of ten Microcystis strains revealed that the different genotypes share a highly similar core genome, while their biosynthetic gene clusters (BGCs) involved in natural product (NP) formation show a sporadic distribution. Moreover, we uncovered three cryptic BGCs not

Developmental or metabolic changes in chloroplasts can have profound effects on the rest of the plant cell. Such intracellular responses are associated with signals that originate in chloroplasts and convey information on their physiological status to the nucleus, which leads to large-scale changes in gene expression (retrograde signaling). A screen designed to identify components of retrograde signaling resulted in the discovery of the so-called genomes uncoupled (gun) mutants. Genetic evidence suggests that the chloroplast protein GUN1 integrates signals derived from perturbations in plastid redox state, plastid gene expression, and tetrapyrrole biosynthesis (TPB) in Arabidopsis (Arabidopsis thaliana) seedlings, exerting biogenic control of chloroplast functions. However, the molecular mechanism by which GUN1 integrates retrograde signaling in the chloroplast is unclear. Here we show that GUN1 also operates in adult plants, contributing to operational control of chloroplasts. The gun1 mutation genetically interacts with mutations of genes for the chloroplast ribosomal proteins S1 (PRPS1) and L11. Analysis of gun1 prps1 lines indicates that GUN1 controls PRPS1 accumulation at the protein level. The GUN1 protein physically interacts with proteins involved in chloroplast protein homeostasis based on coimmunoprecipitation experiments. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments suggest that GUN1 might transiently interact with several TPB enzymes, including Mg-chelatase subunit D (CHLD) and two other TPB enzymes known to activate retrograde signaling. Moreover, the association of PRPS1 and CHLD with protein complexes is modulated by GUN1. These findings allow us to speculate that retrograde signaling might involve GUN1-dependent formation of protein complexes.

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.

A eukaryote-type actin and its binding protein profilin encoded on a genomic island in the cyanobacterium Microcystis aeruginosa PCC 7806 co-localize to form a hollow, spherical enclosure occupying a considerable intracellular space as shown by in vivo fluorescence microscopy. Biochemical and biophysical characterization reveals key differences between these proteins and their eukaryotic homologs. Small-angle X-ray scattering shows that the actin assembles into elongated, filamentous polymers which can be visualized microscopically with fluorescent phalloidin. Whereas rabbit actin forms thin cylindrical filaments about 100 mu m in length, cyanobacterial actin polymers resemble a ribbon, arrest polymerization at 510 lam and tend to form irregular multi-strand assemblies. While eukaryotic profilin is a specific actin monomer binding protein, cyanobacterial profilin shows the unprecedented property of decorating actin filaments. Electron micrographs show that cyanobacterial profilin stimulates actin filament bundling and stabilizes their lateral alignment into heteropolymeric sheets from which the observed hollow enclosure may be formed. We hypothesize that adaptation to the confined space of a bacterial cell devoid of binding proteins usually regulating actin polymerization in eukaryotes has driven the co-evolution of cyanobacterial actin and profilin, giving rise to an intracellular entity.

International audience; The concept of horizontal gene transfer (HGT) as a potent evolutionary force has prompted the re-evaluation of prokaryotic genome shaping and speciation. Horizontal gene transfer enables prokaryotes to rearrange their genomes dynamically, facilitating responses to changing environmental conditions and invasions of new ecological niches. Here we report that the genome of the cyanobacterium Microcystis aeruginosa contains a genomic island encoding proteins with extensive amino-acid sequence identity to two components of the eukaryotic actin cytoskeleton: actin itself; and profilin, an actin binding protein hitherto only known in eukaryotes. Our data indicate that a rare eukaryote-to-prokaryote HGT has introduced both sequences into the Microcystis lineage. We found both genes to be actively expressed and propose a unique role in Microcystis cell stabilization for actin, differing substantially from what is observed for bacterial actin homologs. Because we detected both eukaryote-like genes only in one strain in culture and in recent samples collected from its original habitat we suggest that both proteins may contribute to the adaptation of this strain to its specific ecological niche.