Research for biotechnological applications of cyanobacteria focuses on synthetic pathways and bioreactor design, while little effort is devoted to introduce new, promising organisms in the field. Applications are most often based on recombinant work, and the establishment of transformation can be a risky, time-consuming procedure. In this work we demonstrate the natural transformation of the filamentous cyanobacterium Phormidium lacuna and insertion of a selection marker into the genome by homologous recombination. This is the first example for natural transformation filamentous non-heterocystous cyanobacterium. We found that Phormidium lacuna is polyploid, each cell has about 20-90 chromosomes. Transformed filaments were resistant against up to 14 mg/ml of kanamycin. Formerly, natural transformation in cyanobacteria has been considered a rare and exclusive feature of a few unicellular species. Our finding suggests that natural competence is more distributed among cyanobacteria than previously thought. This is supported by bioinformatic analyses which show that all protein factors for natural transformation are present in the majority of the analyzed cyanobacteria.

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9Research for biotechnological applications of cyanobacteria focuses on synthetic pathways and 10 bioreactor design, while little effort is devoted to introduce new, promising organisms in the field. 11Applications are most often based on recombinant work, and the establishment of transformation 12 can be a risky, time-consuming procedure. In this work we demonstrate the natural transformation 13 of the filamentous cyanobacterium Phormidium lacuna and insertion of a selection marker into the 14 genome by homologous integration. This is the first example for natural transformation of a member 15 of the order Oscillatoriales. We found that Phormidium lacuna is polyploid, each cell has about 20-16 100 chromosomes. Transformed filaments were resistant against up to 15 mg/ml of kanamycin, and 17 the high resistance feature allowed for rapid segregation into all chromosomes. Formerly, natural 18 transformation in cyanobacteria has been considered a rare and exclusive feature of a few unicellular 19 species. Our finding suggests that natural competence is more distributed among cyanobacteria than 20 previously thought. This is supported by bioinformatic analyses which show that all protein factors 21 for natural transformation are present in the majority of the analyzed cyanobacteria. 22

17Lateral DNA transfer plays an important role in the evolution of genetic diversity in 18 prokaryotes. DNA acquisition via transformation involves the uptake of DNA from the 19 environment. The ability of recipient cells to actively transport DNA into the cytoplasm -20 termed natural competencedepends on the presence of type IV pili and competence 21 proteins. Natural competence has been described in cyanobacteria for several organisms 22 including unicellular and filamentous species. However, the presence of natural competence 23 in ramified cyanobacteria, which are considered the peak of cyanobacterial morphological 24 complexity, remains unknown. Here we show that ramified cyanobacteria harbour the genes 25 essential for natural competence and experimentally demonstrate natural competence in the 26 ramified cyanobacterium Chlorogloeopsis fritschii PCC 6912 (hereafter Chlorogloeopsis). 27Searching for homologs to known natural competence genes in ramified cyanobacteria 28 showed that these genes are conserved in the majority of tested isolates. Experimental 29 validation of natural competence using several alternative protocols demonstrates that 30Chlorogloeopsis could be naturally transformed with a replicative plasmid. Our results show 31 that natural competence is a common trait in ramified cyanobacteria and that natural 32 transformation is likely to play an important role in cyanobacteria evolution. 33 34 Importance Cyanobacteria are crucial players in the global biogeochemical cycles where 35 they contribute to CO 2 -and N 2 -fixation. Their main ecological significance is the oxygen-36 producing photosynthetic apparatus that contributes to contemporary food chains. Ramified 37 cyanobacteria form true-branching and multiseriate cell filament structures that represent a 38 peak of prokaryotic multicellularity. Species in that group inhabit fresh and marine water 39 habitats, thermal springs, arid environments, as well as endolithic and epiphytic habitats. 40Here we show that ramified cyanobacteria harbor the mechanisms required for DNA 41 acquisition via natural transformation. The prevalence of mechanisms for natural uptake of 42 DNA has implications for the role of DNA acquisition in the evolution of cyanobacteria. 43Furthermore, presence of mechanisms for natural transformation in ramified cyanobacteria 44 opens up new possibilities for genetic modification of ramified cyanobacteria. 45 46

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