Engineering natural competence into the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973.

Natural transformation is the process by which bacteria actively take up and integrate extracellular DNA into their genomes. In cyanobacteria, natural transformation has only been experimentally demonstrated in a handful of species. Although, cyanobacteria are important model systems for studying photosynthesis and circadian cycling, natural transformation in cyanobacteria has not been characterized to the degree that the process has been studied in other gram-negative bacteria. Two cyanobacterial species that are 99.8% genetically identical provide a unique opportunity to better understand the nuances of natural transformation in cyanobacteria: Synechococcus elongatus PCC 7942 and Synechococcus elongatus UTEX 2973 (hereafter Synechococcus 7942 and Synechococcus 2973 respectively). Synechococcus 7942 is a naturally transformable model system, while Synechococcus 2973 is a recently discovered species that is not naturally competent. Taking only 1.5 hours to replicate, Synechococcus 2973 is the fastest growing cyanobacterial species known, and thus is a strong candidate for serving as a model organism. However, the organism's inability to undergo natural transformation has prevented it from becoming a widely used model system. By substituting polymorphic alleles from Synechococcus 7942 for native Synechococcus 2973 alleles, natural transformation was introduced into Synechococcus 2973. Two genetic loci were found to be involved in differential natural competence between the two organisms: transformation pilus component pilN and circadian transcriptional master regulator rpaA. By using targeting genome editing and enrichment outgrowth, a strain that was both naturally transformable and fast-growing was created. This new Synechococcus 2973-T strain will serve as a valuable resource to the cyanobacterial research community. Importance Certain bacterial species have the ability to take up naked extracellular DNA and integrate it into their genomes. This process is known as natural transformation and is widely considered to play a major role in bacterial evolution. Because of the ease of introducing new genes into naturally transformable organisms, this capacity is also highly valued in the laboratory. Cyanobacteria are photosynthetic and can therefore serve as model systems for some important aspects of plant physiology. Here, we describe the creation of a modified cyanobacterial strain (Synechococcus 2973-T) that is capable of undergoing natural transformation and has a replication time that is on par with the fastest-growing cyanobacterium that has been discovered to date. This new cyanobacterium has the potential to serve as a new model organism for the cyanobacterial research community and will allow experiments to be completed in a fraction of the time that it took to complete previous assays.