Toward a genetic system in the marine cyanobacteriumProchlorococcus

As the smallest and most abundant primary producer in the oceans, the cyanobacteriumProchlorococcusis of interest to diverse branches of science. For the past 30 years, research on this minimal phototroph has led to a growing understanding of biological organization across multiple scales, from the genome to the global ocean ecosystem. Progress in understanding drivers of its diversity and ecology, as well as molecular mechanisms underpinning its streamlined simplicity, has been hampered by the inability to manipulate these cells genetically.Multiple attempts have been made to develop an efficient genetic transformation method forProchlorococcusover the years; all have been unsuccessful to date, despite some success with their close relative,Synechococcus. To avoid the pursuit of unproductive paths, we report here what has not worked in our hands, as well as our progress developing a method to screen the most efficient electroporation parameters for optimal DNA delivery intoProchlorococcuscells. We also report a novel protocol for obtaining axenic colonies and a new method for differentiating live and dead cells. The electroporation method can be used to optimize DNA delivery into any bacterium, making it a useful tool for advancing transformation systems in other genetically recalcitrant microorganisms.